Shatterproof genes and mutations

ABSTRACT

The present disclosure provides shatterproof (SHP) genes and plants and/or plant cells bearing one or more mutations in a shatterproof gene; as well as methods of making and using such plants. In some embodiments the plant or plant cell is resistant to preharvest dehiscence.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation application of U.S. National Stage application Ser. No. 16/959,331, filed on Jun. 30, 2020, which is a National Stage Application under 35 U.S.C. § 371 of International Application No. PCT/US2019/012938, internationally filed on Jan. 9, 2019, which claims the benefit of U.S. Provisional Application No. 62/615,409, filed on Jan. 9, 2018, and U.S. Provisional Application No. 62/732,397, filed on Sep. 17, 2018, each of which are incorporated herein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 165072000101SEQLIST.TXT, date recorded: Apr. 20, 2022, size: 92,047 bytes).

FIELD

The present disclosure relates to compositions and methods pertaining to novel plant genes and gene products and also to plants having one or more gene mutations. In particular, the present disclosure provides shatterproof (SHP) genes and plants and/or plant cells bearing one or more mutations in a shatterproof gene; as well as methods of making and using such plants. In some embodiments the plant or plant cell is resistant to preharvest dehiscence.

BACKGROUND

Preharvest dehiscence of canola seed pods is a process of agronomic importance that causes significant yield loss as well as carry over of a crop into the subsequent growing season. Accordingly, there exists a need for improved methods of reducing or preventing preharvest dehiscence of seed pods, as well as for improved plants that exhibit improved resistance to or reduced susceptibility to preharvest dehiscence.

BRIEF SUMMARY

The present disclosure is based at least in part on the discovery that Brassica plants have eight shatterproof genes; and that causing mutations to one or more of such genes can reduce preharvest dehiscence in agriculture crops such as Brassica crops.

A shatterproof (SHP) gene as used herein means a gene having a sequence as represented by the Brassica napus SHP1A, SHP1C, SHP2A, SHP2C, SHP3A, SHP3C, SHP4A, SHP4C sequences as disclosed herein or in certain embodiments, homologs, variants or mutants thereof. The term “shatterproof homolog” or any variation refers to a shatterproof gene or shatterproof gene product found in another species that performs the same or substantially the same biological function as the Brassica genes and gene products disclosed herein and where the nucleic acid sequences of the coding region or polypeptide sequences (of the SHP gene product) are said to be “identical” or at least 50%, or at least 60%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98% or at least 99% similar (also referred to as “percent identity” or “substantially identical”) to one or more of SHP1A, SHP1C, SHP2A, SHP2C, SHP3A, SHP3C, SHP4A, SHP4C sequences as disclosed herein.

In a first aspect, provided is a method of preventing or reducing preharvest dehiscence in a plant, said method comprising mutating at least one endogenous shatterproof gene in a cell of said plant. In some embodiments the method includes (1) introducing into plant cells a gene repair oligonucleobase to produce plant cells with a mutant SHP gene; and (2) regenerating a non-transgenic plant having a mutated SHP gene from said selected plant cell. In some embodiments the method includes (1) introducing into plant cells a DNA cutter configured to specifically nick or cut a SHP gene to produce plant cells with a mutant SHP gene; and (2) regenerating a non-transgenic plant having a mutated SHP gene from said selected plant cell. In a related embodiment, provided is method comprising contacting a cell with a DNA cutter configured to specifically nick or cut a shatterproof gene. In a related aspect, provided are methods of making a mutation in a SHP gene. In some embodiments the method or methods as described herein may include exposing the cell to a DNA cutter and a GRON. In certain embodiments the methods include exposing a cell to a DNA cutter and a GRON wherein said GRON is modified with one or more of a Cy3 group, 3PS group, and a 2′O-methyl group. In some embodiments the method or methods may include exposing the cell to a DNA cutter without exposing the cell to a GRON. In some embodiments that include exposure to a DNA cutter, the DNA cutter specifically targets a SHP gene. In some embodiments the DNA cutter is one or more selected from a CRISPR which includes but is not limited to Cas9, Cpf1 and their corresponding homologues, orthologues and/or paralogues, a base editor, a TALEN, a zinc finger, meganuclease, and a DNA-cutting antibiotic. In some embodiments the DNA cutter can be plasmid (DNA), RNA and/or protein. In certain embodiments, the methods provided do not include contacting the plant or plant cell with any transgene. In some embodiments of any of the aspects and embodiments provided herein, the plant or plant cell is non-transgenic. In certain aspects, the mutation, alteration or modification to a SHP gene includes an insertion or deletion. In some embodiments the mutation, alteration or modification is or includes a nucleotide change or substitution. In some embodiments of the method, the alteration, mutation or modification introduces a premature stop codon. In some embodiments the alteration, mutation or modification introduces a frame shift mutation. In some embodiments of the compositions and methods provided herein, the mutation relative to a wildtype a SHP gene is an +1, −1, −2 nucleotide insertion or deletion (InDel). In certain embodiments of the compositions and methods provided herein, the mutation relative to a wildtype a SHP gene is an +1, −1, −2 nucleotide insertion or deletion (InDel) developed by a targeted mutation. In some embodiments of the methods provided herein, the mutation, modification or alteration in the SHP gene reduces or obviates the activity or expression of the SHP gene. In certain embodiments of the methods provided herein, at least one SHP gene; or at least two SHP genes; or at least three SHP genes; or at least four SHP genes; or at least five SHP genes; or at least six SHP genes; or at least seven SHP genes; or eight SHP genes are modified. In certain aspects, the mutation, alteration or modification includes an insertion or deletion. In some embodiments the mutation, alteration or modification includes a nucleotide change or substitution. In some embodiments of the method, the alteration, mutation or modification introduces a premature stop codon. In some embodiments of the methods provided herein, the mutation, modification or alteration in the SHP gene reduces or obviates the activity or expression of the SHP gene. In some embodiments, the plant or plant cell is a Brassica plant. In certain embodiments, provided is a plant or plant cell generated by the methods disclosed herein.

In one aspect provided is an isolated nucleic acid the sequence of SHP1A, SHP1C, SHP2A, SHP2C, SHP3A, SHP3C, SHP4A, or SHP4C as disclosed herein or a fragment thereof. In some embodiments, a fragment of one or more of the aforementioned SHP gene sequences includes at least 80%; or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98% or at least 99% of the entire sequence of the gene. In a related aspect, provided is an isolated amino acid sequence encoded by a SHP1A, SHP1C, SHP2A, SHP2C, SHP3A, SHP3C, SHP4A, or SHP4C nucleic acid sequence as disclosed herein or a fragment thereof.

In another aspect, provided is a plant or plant cell having at least three, or at least four, or at least five, or at least seven, or eight shatterproof genes having a sequence that is different than any naturally occurring shatterproof gene.

In one aspect, provided is a plant or plant cell having at least three, or at least four, or at least five, or at least seven, or eight endogenous shatterproof genes having a sequence that is different than any naturally occurring shatterproof gene.

In another aspect, provided is a canola plant or canola plant cell having at least one, or at least two, or at least three, or at least four, or at least five, or at least seven, or eight shatterproof genes having a sequence that is different than any naturally occurring shatterproof gene.

In certain aspects and embodiments, it is desirable to have dehiscence to occur (although not prematurely) and, thus, it may in some embodiments to retain a certain amount of activity of a gene product of one or more of the SHP1A, SHP1C, SHP2A, SHP2C, SHP3A, SHP3C, SHP4A, or SHP4C genes/loci. Accordingly, in one embodiment, provided is a plant or plant cell having three to seven SHP genes having a sequence that is different than any naturally occurring shatterproof gene. In another embodiment, provided is a plant or plant cell having three to six SHP genes having a sequence that is different than any naturally occurring shatterproof gene. In another embodiment, provided is a plant or plant cell having three to five SHP genes having a sequence that is different than any naturally occurring shatterproof gene. In another embodiment, provided is a plant or plant cell having four to six SHP genes having a sequence that is different than any naturally occurring shatterproof gene. In another embodiment, provided is a plant or plant cell having four or five SHP genes having a sequence that is different than any naturally occurring shatterproof gene. In another embodiment, provided is a plant or plant cell having three or four SHP genes having a sequence that is different than any naturally occurring shatterproof gene. In another embodiment, provided is a plant or plant cell having three SHP genes having a sequence that is different than any naturally occurring shatterproof gene. In another embodiment, provided is a plant or plant cell having four SHP genes having a sequence that is different than any naturally occurring shatterproof gene. In another embodiment, provided is a plant or plant cell having five SHP genes having a sequence that is different than any naturally occurring shatterproof gene. In another embodiment, provided is a plant or plant cell having six SHP genes having a sequence that is different than any naturally occurring shatterproof gene. In another embodiment, provided is a plant or plant cell having seven SHP genes having a sequence that is different than any naturally occurring shatterproof gene.

In a certain aspect, provided is a plant or plant cell having a mutation in a SHP1A, SHP1C, SHP2A, SHP2C, SHP3A, SHP3C, SHP4A, or SHP4C gene. In some embodiments of this aspect, the SHP gene is an endogenous SHP gene.

In another aspect, the present disclosure relates to a plant or part thereof including at least one mutation in at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight nucleic acid sequences encoding SHATTERPROOF (SHP) genes. In some embodiments, the nucleic acid sequences have at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to nucleic acid sequences selected from the group of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. In some embodiments, the nucleic acid sequences are selected from the group of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. In some embodiments that may be combined with any of the preceding embodiments, the mutation is a frameshift mutation. In some embodiments, the frameshift mutation results in one or more nucleotide insertions or deletions as compared to the corresponding endogenous gene without the frameshift mutation. In some embodiments that may be combined with any of the preceding embodiments, the frameshift mutation results in a premature stop codon. In some embodiments that may be combined with any of the preceding embodiments, the mutation reduces or eliminates expression of the SHP gene and/or SHP polypeptide. In some embodiments that may be combined with any of the preceding embodiments, the plant exhibits reduced susceptibility to preharvest dehiscence. In some embodiments that may be combined with any of the preceding embodiments, the plant is selected from the group of Brassica napus, Brassica rapa, Brassica oleracea, Brassica juncea, Brassica species, Raphanus sativus, Pisum sativum, Phaseolus vulgaris, Lens culinaris, Glycine max, and Fabaceae species.

In another aspect, the present disclosure relates to a method of producing the plant or part thereof of any of the preceding embodiments, including the steps of: a) introducing mutations into plant cells, wherein the mutations are at least one mutation in at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight nucleic acid sequences encoding SHP genes; b) selecting plant cells containing the mutations; and c) regenerating a plant having the mutations; wherein the plant exhibits reduced susceptibility to preharvest dehiscence. In some embodiments, wherein the mutations are introduced using one or more vectors, wherein the vectors include gene editing components selected from the group of a CRISPR/Cas9 system, a TALEN, a zinc finger, and a meganuclease designed to target a nucleic acid sequence encoding a SHP gene. In some embodiments, the mutations are introduced using a GRON system designed to target a nucleic acid sequence encoding a SHP gene. In some embodiments, the GRON system comprises one or more modifications selected from the group consisting of a Cy3 group, 3PS group, and a 2′O-methyl group. In some embodiments that may be combined with any of the preceding embodiments, the nucleic acid sequences have at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to nucleic acid sequences selected from the group of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. In some embodiments that may be combined with any of the preceding embodiments, the nucleic acid sequences are selected from the group of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. In some embodiments that may be combined with any of the preceding embodiments, the mutation is selected from the group of a frameshift mutation, a frameshift mutation resulting in one or more nucleotide insertions or deletions as compared to the corresponding endogenous gene without the frameshift mutation, and a frameshift mutation resulting in a premature stop codon, and wherein the mutation reduces or eliminates expression of the SHP gene and/or SHP polypeptide. In some embodiments that may be combined with any of the preceding embodiments, the plant is selected from the group of Brassica napus, Brassica rapa, Brassica oleracea, Brassica juncea, Brassica species, Raphanus sativus, Pisum sativum, Phaseolus vulgaris, Lens culinaris, Glycine max, and Fabaceae species.

In another aspect, the present disclosure provides an F₁ plant, where the F₁ plant has the plant of any one of the preceding embodiments as a parent. In another aspect, the present disclosure provides a method of making plant seeds, the method including crossing the plant of any one of the preceding embodiments with another plant and harvesting seed therefrom. In another aspect, the present disclosure provides a method of making a plant of any one of the preceding embodiments, the method including selecting seeds from the cross of the plant of any one of the preceding embodiments with the plant of any one of the preceding embodiments to make the plant of any one of the preceding embodiments. In another aspect, the present disclosure provides a plant produced by growing the seed of any one of the preceding embodiments, where the plant has all the physiological and morphological characteristics of the plant of any one of the preceding embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the office upon request and payment of the necessary fee.

FIGS. 1A-1B illustrate CLUSTAL multiple sequence alignments of partial nucleotide sequences and deduced amino acid sequences of Brassica napus SHATTERPROOF (BnSHP) genes and Arabidopsis thaliana SHP genes (AtSHP1 and AtSHP2). FIG. 1A illustrates CLUSTAL multiple sequence alignment of partial nucleotide sequences of BnSHP1A (SEQ ID NO: 30), BnSHP1C (SEQ ID NO: 31), BnSHP2A (SEQ ID NO: 23), BnSHP2C (SEQ ID NO: 22), BnSHP3A (SEQ ID NO: 28), BnSHP3C (SEQ ID NO: 29), BnSHP4A (SEQ ID NO: 25), BnSHP4C (SEQ ID NO: 26), AtSHP1 (SEQ ID NO: 24), and AtSHP2 (SEQ ID NO: 27) beginning at the start codon (B. napus nucleotide sequences obtained from gDNA of the BN2-SU line). FIG. 1B illustrates CLUSTAL multiple sequence alignment of deduced amino acid sequences of BnSHP1A (SEQ ID NO: 34), BnSHP1C (SEQ ID NO: 35), BnSHP2A (SEQ ID NO: 36), BnSHP2C (SEQ ID NO: 37), BnSHP3A (SEQ ID NO: 39), BnSHP3C (SEQ ID NO: 38), BnSHP4A (SEQ ID NO: 40), BnSHP4C (SEQ ID NO: 41), AtSHP1 (SEQ ID NO: 32), and AtSHP2 (SEQ ID NO: 33).

FIGS. 2A-2B illustrate SHP gene expression analysis by next generation sequencing (NGS). FIG. 2A illustrates developmental stages of fruit samples taken for SHP gene expression analysis: developmental stage 13=anthesis, when flowers open and self-pollinate; developmental stages 17-1, 17-2, 17-3, 17-4, and 17-5=fruits of increasing sizes during the elongation stage (Roeder and Yanofsky, 2006). FIG. 2B illustrates the percentage of total reads identified for each SHP gene at each fruit developmental stage after RT-PCR and NGS analysis (developmental stages shown from left to right for each SHP gene in the order 13, 17-1, 17-2, 17-3, 17-4, and 17-5).

FIG. 3 illustrates the total percentage of insertions and deletions (InDels) identified by NGS in each of the SHP genes in shoots regenerated from control non-treated protoplasts (SHPA01-1 and SHPA01-2, lighter gray bars shown on left for each SHP gene), and protoplasts treated with CRISPR/Cas9 plasmid (SHPA01-3 and SHPA01-4, darker gray bars shown on right for each SHP gene). Treatments were performed in duplicate.

FIG. 4 illustrates phloroglucinol staining of lignified cell layers in canola pods (siliques). The left panel illustrates a silique (lengthwise image of full silique). Siliques are derived from two carpels that form two locules separated by the septum. The fruit walls are valves containing the seeds that are attached to the replum forming a suture. The top middle panel illustrates a transverse section of the silique valves containing the seeds attached to the septum. The bottom middle panel illustrates a transverse section of the silique valves containing the seeds attached to the septum stained with phloroglucinol, showing lignified cell layers in the region of attachment of the silique valve to the replum and the lignified endocarp-b cell layer. The top right panel illustrates lignified cell layers in a cross section of a wild type silique (phloroglucinol staining score=1). The bottom right panel illustrates the absence of lignified cell layer in a cross section of a pod from a full SHP knock-out (KO) mutant line (phloroglucinol staining score=5). The absence of the lignified cell layer in the region of attachment of the silique valve to the replum has been associated with a high level of shatter resistance in oilseed Brassicas.

FIG. 5 illustrates a pod shattering test of selected KO plants (C₀) with different numbers of BnSHP gene KOs. Dried and undried mature pods were obtained from wild type (0 KO) plants and KO plants with 2, 3, 5, 6, or 7 SHP gene KOs (KOs shown from left to right at each frequency shown in order 0 KO (undried), 0 KO (dried), 2 KO (dried), 3 KO (dried), 5 KO (dried), 6 KO (dried), and 7 KO (dried)).

FIG. 6 illustrates the correlation between the shattering frequency determined with the TissueLyser (Hz) and the shattering frequency determined with the Geno/Grinder (RPM). R value=0.88; p value=0.00016.

FIG. 7 illustrates phenotype and genotype data of shatterproof KO canola lines and checks (controls).

DETAILED DESCRIPTION

Various aspects and embodiments of the present disclosure provide a plant having one or more SHP mutations and/or mutation combinations, methods of making such a plant, and methods for reducing preharvest dehiscence.

One skilled in the art readily appreciates that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure.

It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the disclosure disclosed herein without departing from the scope and spirit of the disclosure.

The disclosure illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed. Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure as defined by the appended claims.

Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification, improvement, and variation of the disclosures disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure.

The disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the disclosure with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

Preharvest Dehiscence

Siliques or pods from Brassica plants release their seeds through a process called fruit dehiscence. Shedding of seed (also referred to as “seed shatter” or “pod shatter”) by mature pods before or during crop harvest is a universal phenomenon with crops that develop dry dehiscent fruits. Premature seed shatter results in a reduced seed recovery, which represents a problem in crops that are grown primarily for the seeds, such as oil-producing Brassica plants, particularly oilseed rape. Another problem related to premature seed shattering is an increase in volunteer (weed) growth in the subsequent crop year.

Preharvest dehiscence of canola seed pods is a process of agronomic importance that causes significant yield loss as well as carryover of a crop into the subsequent growing season. In canola, pod shatter causes an annual yield loss of 20% and may result in losses of 50% when harvest is delayed, and under adverse weather conditions. Seed shattering occurs in ripe standing crops during hot and windy summers due to impact from other plants, and in windrows from the impact of harvest machinery (MacLeod, 1981; Child and Evans, 1989). In real terms, shatter results in a yield loss of $20-$25 per acre ($39M annual yield loss in the US) and swathing costs add an additional $6 per acre ($8.7 M annual additional direct cost; Barry Coleman, Northern Canola Growers Association).

As used herein, the fruit of the Brassicaceae develops from a gynoecium composed of two fused carpels, which, upon fertilization, grow to become a silique with two locules (valves) that contain the developing seeds (See FIG. 4 , Example 4). The fruit walls are the valves that are attached to the replum (the persisting septa of the ovary) forming a suture, also called dehiscence zone (DZ), along the valve margins. The DZ typically consists of a thin layer of parenchyma cells that acts as a separation layer upon fruit ripening, when cell wall degrading enzymes, such as cellulases and polygalaturonases are secreted, reducing cellular cohesion, and predisposing pods to shattering by external mechanical forces (Meakin and Roberts, 1990a,b). The absence of the separation layer in the region of attachment of the silique valve to the replum has been associated with a high level of shatter resistance in oilseed Brassicas (Kadkol et al., 1986; Meakin and Roberts, 1990a,b; Liljegren et al., 2000).

The lignified cell layer, as used herein, refers to another layer of specialized cells along the valve margins that contribute to the opening of the fruit, in addition to the separation layer (See FIG. 4 ). At maturity, the lignified cell layer in the valve and the replum delimit the non-lignified separation layer in the valve margins. The stiffening of cell walls through lignification of the lignified margin layer, and the internal lignified endocarp b valve layer has been proposed to contribute mechanically to fruit opening (Spence et al., 1996). As the fruit dries, differential shrinkage of the remaining thin-walled valve cells relative to the rigid lignified margin and valve layers is thought to create internal tension, causing the shattering that is characteristic of fruit dehiscence.

SHATTERPROOF (SHP), as used herein, refer to transcription factors members of the MADS-box family involved in the differentiation of the DZ in developing pods in the Brassicaceae (Liljegren et al., 2000). Loss-of-function studies indicate that SHP promote cell wall lignification of the valve margin cells (i.e., lignified cell layer) in Arabidopsis fruit. Arabidopsis shp1shp2 double mutants develop a non-functional DZ that does not fully differentiate a layer of cells with lignified cell walls, nor a separation cell layer, and, as a consequence, the fruits are indehiscent and do not open at the end of development (Liljegren et al., 2000). SHP encoding genes are expressed at the valve margins from the early stages of gynoecium development, where they activate the expression of bHLH factors INDEHISCENT (IND), essential for both separation and lignified layer development, and ALCATRAZ (ALC), required only for separation layer formation (Roeder and Yanofsky, 2006).

As used herein, Oilseed Rape (syn. canola, rapeseed, Brassica napus L., spp. oleifera; genomes AACC, 2n=4×=38), also a member of the Brassicaceae, is an allopolyploid plant originated through spontaneous hybridization between turnip rape (Brassica rapa L.; genome AA, 2n=2×=20), and cabbage (Brassica oleracea L.; genome CC, 2n=2×=18) (Chalhoub et al., 2014). Homologs of Arabidopsis SHP1/2 (as well as other functionally-related transcription factors IND and ALC) have been found in canola, and molecular genetic research has previously shown several quantitative trait loci (QTL) associated with shattering with epistatic relationships between them (Gururaj, 2009; Raman et al, 2014).

Increased pod shatter resistance, as used herein, refers to the reduction of seed shattering of mature (dried) fruits, as a consequence of external mechanical forces in the laboratory and in the field. Laboratory tests simulate the process of pod shattering as it occurs under natural field conditions, and the results normally correlate with the field measurements. Field evaluation alone of shatter resistance can be inaccurate due to varying weather conditions during harvest time in different seasons and locations.

As used herein, fruit anatomical characters are associated with pod shatter resistance. Differentiation of the lignified valve margin cells and the separation cell layer determines the level of seed shattering. In Arabidopsis, the loss of phloroglucinol-stainable lignified valve margin cells positively correlates with a higher resistance of the pods to mechanical shatter. Phloroglucinol is a common dye used to stain cell wall lignin in plant tissue. After staining with phloroglucinol, lignified cell walls appear red-violet, and the intensity of the stain (color) positively correlates with the level of lignin deposition and differentiation of the cells. Lignified valve margin cells readily stain with phloroglucinol in cross sections of wild type fruits of Arabidopsis and Oil Seed Rape (OSR). The separation layer cells of the DZ do not contain lignin, and they are not stained with phloroglucinol. The fruits of Arabidopsis shp1shp2 double mutant plants do not differentiate valve margin cells with lignified cell walls, and therefore they are not stained with phloroglucinol (Liljegren et al., 2000).

A pod breaking test, as used herein, refers to a laboratory test that uses a tissue lyser to assess the shatter resistance of mature, fully dried pods. The shatterproof phenotype was determined by the level of valve separation found under controlled agitation of the pods. For this test, single pods are placed in a 96 well deep trough container and secured in the arms of a TissueLyser II (Qiagen, Germany). The single pod samples are run on the TissueLyser for 30 seconds at frequencies of 22, 23, 24, 25, 26, 27, 28, 29, and 30 Hz. Four single pods reps per plant are tested at each frequency. The phenotype is scored on a scale of 1-5 (See e.g. Example 4). An intact pod is given a score of one, a partially split pod with connected valves is scored a two, a score of three represents the separation of one valve, and a score of four indicates that both valves are separated from the replum. A high correlation is found between the shaking frequency to shatter score of dried pods and the phloroglucinol staining score of lignified layers of developing pods (r=0.797). This demonstrated that the lignified layer staining of fruits and the shaking frequency to shatter test could be used to effectively evaluate the shatterproof trait.

Pod shattering could also be determined using the Geno/Grinder 2010 (SPEX Sample Prep, USA). In this case, the shatterproof phenotype is determined by the level of valve separation found under controlled agitation of the pods. To test the valve separation, 12-24 pods are placed into a 96 well deep trough container and secured in the arms of a Geno/Grinder 2010. The containers holding pod samples are run for 20 seconds at different rpm (for example at 720, 750, 780, 810, 840, 870, 900, 930, 960, 990, 1020, 1050, 1080 rpm). At the end of the run, the container is taken off the machine and the shattering score is given to each pod according to the score table (see Example 4). When the average shattering score under the certain rpm is greater than 2.5, the rpm value will be the pod shattering value for the line.

Shatterproof (SHP) Genes

The present disclosure generally relates to plants having mutations in shatterproof (SHP) genes. In some embodiments, one or more mutations in one or more SHP genes results in increased resistance to/reduced susceptibility to preharvest dehiscence.

In some aspects, plants of the present disclosure are Brassica napus L., spp. oleifera (canola, oilseed rape) plants. Canola plants contain eight SHATTERPROOF (SHP) genes, designated BnSHP1A, BnSHP1C, BnSHP2A, BnSHP2C, BnSHP3A, BnSHP3C, BnSHP4A and BnSHP4C. In some aspects, plants of the present disclosure have at least one mutation in at least one SHP gene.

Certain aspects of the present disclosure relate to BnSHP1A. The nucleotide coding sequence of BnSHP1A is set forth in SEQ ID NO: 1. Provided herein are also homologs and orthologs of BnSHP1A. In some embodiments, a homolog or ortholog of BnSHP1A has a nucleic acid coding sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In some embodiments, a nucleic acid sequence encoding a homolog or ortholog of BnSHP1A may also have one or more mutations.

Certain aspects of the present disclosure relate to BnSHP1C. The nucleotide coding sequence of BnSHP1C is set forth in SEQ ID NO: 2. Provided herein are also homologs and orthologs of BnSHP1C. In some embodiments, a homolog or ortholog of BnSHP1C has a nucleic acid coding sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2. In some embodiments, a nucleic acid sequence encoding a homolog or ortholog of BnSHP1C may also have one or more mutations.

Certain aspects of the present disclosure relate to BnSHP2A. The nucleotide coding sequence of BnSHP2A is set forth in SEQ ID NO: 3. Provided herein are also homologs and orthologs of BnSHP2A. In some embodiments, a homolog or ortholog of BnSHP2A has a nucleic acid coding sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3. In some embodiments, a nucleic acid sequence encoding a homolog or ortholog of BnSHP2A may also have one or more mutations.

Certain aspects of the present disclosure relate to BnSHP2C. The nucleotide coding sequence of BnSHP2C is set forth in SEQ ID NO: 4. Provided herein are also homologs and orthologs of BnSHP2C. In some embodiments, a homolog or ortholog of BnSHP2C has a nucleic acid coding sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4. In some embodiments, a nucleic acid sequence encoding a homolog or ortholog of BnSHP2C may also have one or more mutations.

Certain aspects of the present disclosure relate to BnSHP3A. The nucleotide coding sequence of BnSHP3A is set forth in SEQ ID NO: 5. Provided herein are also homologs and orthologs of BnSHP3A. In some embodiments, a homolog or ortholog of BnSHP3A has a nucleic acid coding sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5. In some embodiments, a nucleic acid sequence encoding a homolog or ortholog of BnSHP3A may also have one or more mutations.

Certain aspects of the present disclosure relate to BnSHP3C. The nucleotide coding sequence of BnSHP3C is set forth in SEQ ID NO: 6. Provided herein are also homologs and orthologs of BnSHP3C. In some embodiments, a homolog or ortholog of BnSHP3C has a nucleic acid coding sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6. In some embodiments, a nucleic acid sequence encoding a homolog or ortholog of BnSHP3C may also have one or more mutations.

Certain aspects of the present disclosure relate to BnSHP4A. The nucleotide coding sequence of BnSHP4A is set forth in SEQ ID NO: 7. Provided herein are also homologs and orthologs of BnSHP4A. In some embodiments, a homolog or ortholog of BnSHP4A has a nucleic acid coding sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7. In some embodiments, a nucleic acid sequence encoding a homolog or ortholog of BnSHP4A may also have one or more mutations.

Certain aspects of the present disclosure relate to BnSHP4C. The nucleotide coding sequence of BnSHP4C is set forth in SEQ ID NO: 8. Provided herein are also homologs and orthologs of BnSHP4C. In some embodiments, a homolog or ortholog of BnSHP4C has a nucleic acid coding sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8. In some embodiments, a nucleic acid sequence encoding a homolog or ortholog of BnSHP4C may also have one or more mutations.

In some aspects, plants of the present disclosure have a mutation in BnSHP1A. In some embodiments, these plants may also have mutations in one or more SHP genes selected from BnSHP1C, BnSHP2A, BnSHP2C, BnSHP3A, BnSHP3C, BnSHP4A and BnSHP4C.

In some aspects, plants of the present disclosure have a mutation in BnSHP1C. In some embodiments, these plants may also have mutations in one or more SHP genes selected from BnSHP1A, BnSHP2A, BnSHP2C, BnSHP3A, BnSHP3C, BnSHP4A and BnSHP4C.

In some aspects, plants of the present disclosure have a mutation in BnSHP2A. In some embodiments, these plants may also have mutations in one or more SHP genes selected from BnSHP1A, BnSHP1C, BnSHP2C, BnSHP3A, BnSHP3C, BnSHP4A and BnSHP4C.

In some aspects, plants of the present disclosure have a mutation in BnSHP2C. In some embodiments, these plants may also have mutations in one or more SHP genes selected from BnSHP1A, BnSHP1C, BnSHP2A, BnSHP3A, BnSHP3C, BnSHP4A and BnSHP4C.

In some aspects, plants of the present disclosure have a mutation in BnSHP3A. In some embodiments, these plants may also have mutations in one or more SHP genes selected from BnSHP1A, BnSHP1C, BnSHP2A, BnSHP2C, BnSHP3C, BnSHP4A and BnSHP4C.

In some aspects, plants of the present disclosure have a mutation in BnSHP3C. In some embodiments, these plants may also have mutations in one or more SHP genes selected from BnSHP1A, BnSHP1C, BnSHP2A, BnSHP2C, BnSHP3A, BnSHP4A and BnSHP4C.

In some aspects, plants of the present disclosure have a mutation in BnSHP4A. In some embodiments, these plants may also have mutations in one or more SHP genes selected from BnSHP1A, BnSHP1C, BnSHP2A, BnSHP2C, BnSHP3A, BnSHP3C and BnSHP4C.

In some aspects, plants of the present disclosure have a mutation in BnSHP4C. In some embodiments, these plants may also have mutations in one or more SHP genes selected from BnSHP1A, BnSHP1C, BnSHP2A, BnSHP2C, BnSHP3A, BnSHP3C and BnSHP4A.

In some aspects, plants of the present disclosure have a mutation in at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight of the SHP genes. In some aspects, plants of the present disclosure have a mutation in at least five, at least six, at least seven, or eight of the SHP genes.

In some aspects, the mutation may be a frameshift mutation, a frameshift mutation resulting in one or more nucleotide insertions or deletions as compared to the corresponding endogenous gene without the frameshift mutation, or a frameshift mutation resulting in a premature stop codon, wherein the mutation reduces or eliminates expression of the SHP gene and/or SHP polypeptide.

Methods of Identifying Sequence Similarity

Two polynucleotides or polypeptides are identical if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms “identical” or “percent identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. For polypeptides where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4: 11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).

The phrases “substantially identical,” and “percent identity” in the context of two nucleic acids or polypeptides, refer to sequences or subsequences that have at least 50%, advantageously 60%, preferably 70%, more preferably 80%, and most preferably 90-95% nucleotide or amino acid residue identity when aligned for maximum correspondence over a comparison window as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence.

One of skill in the art will recognize that two polypeptides can also be “substantially identical” if the two polypeptides are immunologically similar. Thus, overall protein structure may be similar while the primary structure of the two polypeptides displays significant variation. Therefore, a method to measure whether two polypeptides are substantially identical involves measuring the binding of monoclonal or polyclonal antibodies to each polypeptide. Two polypeptides are substantially identical if the antibodies specific for a first polypeptide bind to a second polypeptide with an affinity of at least one third of the affinity for the first polypeptide. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence relative to the reference sequence, based on the designated program parameters.

The percentage of “sequence similarity” is the percentage of amino acids or nucleotides which is either identical or changed viz. “sequence similarity”=percent sequence identity)+percent changes). Thus, whenever the term sequence “similarity” is used it embraces sequence “identity” and “changes” to the sequence at some percentage. In certain embodiments, the changes in a sequence permitted by the referenced percent sequence identity are all or nearly all conservative changes; that is, in those embodiments when a sequence is 90% identical, the remaining 10% are all or nearly all conservative changes. The term “nearly all” in this context refers to at least 75% of the permitted sequence changes are conservative changes, more preferably at least 85%, still more preferably at least 90%, and most preferably at least 95%.

Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, 0.4dv. Appl. Math. 2:482 (I 98 I), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 5 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), by software for alignments such as VECTOR NTI Version #11.5 by Life Technologies, Carlsbad, Calif., USA, by the procedures described in ClustalW, Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680 or by visual inspection (see generally, Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 33 89-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (worldwide web address: ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)). In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

Nucleic Acids and Delivery Thereof to Cells

Certain aspects of the present disclosure involve nucleic acids (e.g. SHP genes), as well as nucleic acids having one or more mutations. Various methods exist for inducing mutations in a nucleic acid, as described herein. In some embodiments, one or more nucleic acids may be delivered to a cell, as described herein.

Oligonucleobases

As used herein, an “oligonucleobase” is a polymer of nucleobases, which polymer can hybridize by Watson-Crick base pairing to a DNA having the complementary sequence.

Nucleobases comprise a base, which may be a purine, pyrimidine, or a derivative or analog thereof. Nucleobases include peptide nucleobases, the subunits of peptide nucleic acids, and morpholine nucleobases as well as nucleosides and nucleotides. Nucleosides are nucleobases that contain a pentosefuranosyl moiety, e.g., an optionally substituted riboside or 2′-deoxyriboside. Nucleosides can be linked by one of several linkage moieties, which may or may not contain phosphorus. Nucleosides that are linked by unsubstituted phosphodiester linkages are termed nucleotides.

An oligonucleobase chain may have a single 5′ and 3′ terminus, which are the ultimate nucleobases of the polymer. A particular oligonucleobase chain can contain nucleobases of all types. An oligonucleobase compound is a compound comprising one or more oligonucleobase chains that are complementary and hybridized by Watson-Crick base pairing. Nucleobases are either deoxyribo-type or ribo-type. Ribo-type nucleobases are pentosefuranosyl containing nucleobases wherein the 2′ carbon is a methylene substituted with a hydroxyl, alkyloxy or halogen. Deoxyribo-type nucleobases are nucleobases other than ribo-type nucleobases and include all nucleobases that do not contain a pentosefuranosyl moiety.

An oligonucleobase strand generically includes both oligonucleobase chains and segments or regions of oligonucleobase chains. An oligonucleobase strand has a 3′ end and a 5′ end. When an oligonucleobase strand is coextensive with a chain, the 3′ and 5′ ends of the strand are also 3′ and 5′ termini of the chain.

The oligonucleobase can be introduced into a plant cell using any method commonly used in the art, including but not limited to, microcarriers (biolistic delivery), microfibers (whiskers), electroporation, nucleofection, PEG-mediated delivery, direct DNA uptake and microinjection. Illustrative examples of an oligonucleobase are described below.

The description can be practiced with oligonucleobases having the conformations and chemistries described in the Kmiec I and Kmiec II patents which are incorporated herein by reference. Kmiec I teaches a method for introducing specific genetic alterations into a target gene. The oligonucleobases in Kmiec I and/or Kmiec II contain two complementary strands, one of which contains at least one segment of RNA-type nucleotides (an “RNA segment”) that are base paired to DNA-type nucleotides of the other strand.

Kmiec II discloses that purine and pyrimidine base-containing non-nucleotides can be substituted for nucleotides. U.S. Pat. Nos. 5,756,325; 5,871,984; 5,760,012; 5,888,983; 5,795,972; 5,780,296; 5,945,339; 6,004,804; and 6,010,907 and in International Patent No. PCT/US00/23457; and in International Patent Publication Nos. WO 98/49350; WO 99/07865; WO 99/58723; WO 99/58702; WO 99/40789; U.S. Pat. No. 6,870,075; and US Published Patent Application 20030084473, which are each hereby incorporated in their entirety, disclose additional molecules that can be used for the present description. The term “oligonucleobase” is used herein to denote the molecules that can be used in the methods of the present disclosure and include mixed duplex oligonucleotides, non-nucleotide containing molecules taught in Kmiec II, single stranded oligodeoxynucleotides and other molecules taught in the above noted patents and patent publications.

In one embodiment, the oligonucleobase is a mixed duplex oligonucleotide in which the RNA-type nucleotides of the mixed duplex oligonucleotide are made RNase resistant by replacing the 2′-hydroxyl with a fluoro, chloro or bromo functionality or by placing a substituent on the 2′-O. Suitable substituents include the substituents taught by the Kmiec II. Alternative substituents include the substituents taught by U.S. Pat. No. 5,334,711 (Sproat) and the substituents taught by patent publications EP 629 387 and EP 679 657 (collectively, the Martin Applications), which are incorporated herein by reference. As used herein, a 2′-fluoro, chloro or bromo derivative of a ribonucleotide or a ribonucleotide having a 2′-OH substituted with a substituent described in the Martin Applications or Sproat is termed a “2′-substituted ribonucleotide.” As used herein the term “RNA-type nucleotide” means a 2′-hydroxyl or 2′-substituted nucleotide that is linked to other nucleotides of a mixed duplex oligonucleotide by an unsubstituted phosphodiester linkage or any of the non-natural linkages taught by Kmiec I or Kmiec II. As used herein the term “deoxyribo-type nucleotide” means a nucleotide having a 2′-H, which can be linked to other nucleotides of a MDON by an unsubstituted phosphodiester linkage or any of the non-natural linkages taught by Kmiec I or Kmiec II.

In one embodiment of the present disclosure, the oligonucleobase or GRON is a mixed duplex oligonucleotide that is linked solely by unsubstituted phosphodiester bonds. In alternative embodiments, the linkage is by substituted phosphodiesters, phosphodiester derivatives and non-phosphorus-based linkages as taught by Kmiec II. In yet another embodiment, each RNA-type nucleotide in the mixed duplex oligonucleotide is a 2′-substituted nucleotide. Particularly preferred embodiments of 2′-substituted ribonucleotides are 2′-fluoro, 2′-methoxy, 2′-propyloxy, 2′-allyloxy, 2′-hydroxylethyloxy, 2′-methoxyethyloxy, 2′-fluoropropyloxy and 2′-trifluoropropyloxy substituted ribonucleotides. More preferred embodiments of 2′-substituted ribonucleotides are 2′-fluoro, 2′-methoxy, 2′-methoxyethyloxy, and 2′-allyloxy substituted nucleotides. In another embodiment the mixed duplex oligonucleotide is linked by unsubstituted phosphodiester bonds.

Although mixed duplex oligonucleotide having only a single type of 2′-substituted RNA-type nucleotide is more conveniently synthesized, the methods of the disclosure can be practiced with mixed duplex oligonucleotides having two or more types of RNA-type nucleotides. The function of an RNA segment may not be affected by an interruption caused by the introduction of a deoxynucleotide between two RNA-type trinucleotides, accordingly, the term RNA segment encompasses such an “interrupted RNA segment.” An uninterrupted RNA segment is termed a contiguous RNA segment. In an alternative embodiment an RNA segment can contain alternating RNase-resistant and unsubstituted 2′-OH nucleotides. The mixed duplex oligonucleotides preferably have fewer than 100 nucleotides and more preferably fewer than 85 nucleotides, but more than 50 nucleotides. The first and second strands are Watson-Crick base paired. In one embodiment the strands of the mixed duplex oligonucleotide are covalently bonded by a linker, such as a single stranded hexa, penta or tetranucleotide so that the first and second strands are segments of a single oligonucleotide chain having a single 3′ and a single 5′ end. The 3′ and 5′ ends can be protected by the addition of a “hairpin cap” whereby the 3′ and 5′ terminal nucleotides are Watson-Crick paired to adjacent nucleotides. A second hairpin cap can, additionally, be placed at the junction between the first and second strands distant from the 3′ and 5′ ends, so that the Watson-Crick pairing between the first and second strands is stabilized.

The first and second strands contain two regions that are homologous with two fragments of the target SHP gene, i.e., have the same sequence as the target gene. A homologous region contains the nucleotides of an RNA segment and may contain one or more DNA-type nucleotides of connecting DNA segment and may also contain DNA-type nucleotides that are not within the intervening DNA segment. The two regions of homology are separated by, and each is adjacent to, a region having a sequence that differs from the sequence of the target gene, termed a “heterologous region.” The heterologous region can contain one, two or three mismatched nucleotides. The mismatched nucleotides can be contiguous or alternatively can be separated by one or two nucleotides that are homologous with the target gene. Alternatively, the heterologous region can also contain an insertion or one, two, three or of five or fewer nucleotides. Alternatively, the sequence of the mixed duplex oligonucleotide may differ from the sequence of the target gene only by the deletion of one, two, three, or five or fewer nucleotides from the mixed duplex oligonucleotide. The length and position of the heterologous region is, in this case, deemed to be the length of the deletion, even though no nucleotides of the mixed duplex oligonucleotide are within the heterologous region. The distance between the fragments of the target gene that are complementary to the two homologous regions is identically the length of the heterologous region when a substitution or substitutions is intended. When the heterologous region contains an insertion, the homologous regions are thereby separated in the mixed duplex oligonucleotide farther than their complementary homologous fragments are in the gene, and the converse is applicable when the heterologous region encodes a deletion.

The RNA segments of the mixed duplex oligonucleotides are each a part of a homologous region, i.e., a region that is identical in sequence to a fragment of the target gene, which segments together preferably contain at least 13 RNA-type nucleotides and preferably from 16 to 25 RNA-type nucleotides or yet more preferably 18-22 RNA-type nucleotides or most preferably 20 nucleotides. In one embodiment, RNA segments of the homology regions are separated by and adjacent to, i.e., “connected by” an intervening DNA segment. In one embodiment, each nucleotide of the heterologous region is a nucleotide of the intervening DNA segment. An intervening DNA segment that contains the heterologous region of a mixed duplex oligonucleotide is termed a “mutator segment.”

The change to be introduced into the target gene is encoded by the heterologous region. The change to be introduced into the SHP gene may be a change in one or more bases of the target gene sequence that changes the native amino acid in that position to the desired amino acid.

In another embodiment of the present disclosure, the oligonucleobase is a single stranded oligodeoxynucleotide mutational vector or SSOMV, which is disclosed in International Patent Application PCT/US00/23457, which is incorporated herein by reference in its entirety. The sequence of the SSOMV is based on the same principles as the mutational vectors described in U.S. Pat. Nos. 5,756,325; 5,871,984; 5,760,012; 5,888,983; 5,795,972; 5,780,296; 5,945,339; 6,004,804; and 6,010,907 and in International Publication Nos. WO 98/49350; WO 99/07865; WO 99/58723; WO 99/58702; WO 99/40789; U.S. Pat. No. 6,870,075; and US Published Patent Application 20030084473. The sequence of the SSOMV contains two regions that are homologous with the target sequence separated by a region that contains the desired genetic alteration termed the mutator region. The mutator region can have a sequence that is the same length as the sequence that separates the homologous regions in the target sequence, but having a different sequence. Such a mutator region will cause a substitution.

The nucleotides of the SSOMV are deoxyribonucleotides that are linked by unmodified phosphodiester bonds except that the 3′ terminal and/or 5′ terminal internucleotide linkage or alternatively the two 3′ terminal and/or 5′ terminal internucleotide linkages can be a phosphorothioate or phosphoamidate. As used herein an internucleotide linkage is the linkage between nucleotides of the SSOMV and does not include the linkage between the 3′ end nucleotide or 5′ end nucleotide and a blocking substituent, see supra. In a specific embodiment the length of the SSOMV is between 21 and 55 deoxynucleotides and the lengths of the homology regions are, accordingly, a total length of at least 20 deoxynucleotides and at least two homology regions should each have lengths of at least 8 deoxynucleotides.

The SSOMV can be designed to be complementary to either the coding or the non-coding strand of the target gene. When the desired mutation is a substitution of a single base, it is preferred that both the mutator nucleotides be a pyrimidine. To the extent that is consistent with achieving the desired functional result it is preferred that both the mutator nucleotide and the targeted nucleotide in the complementary strand be pyrimidines. Particularly preferred are SSOMV that encode transversion mutations, i.e., a C or T mutator nucleotide is mismatched, respectively, with a C or T nucleotide in the complementary strand.

In addition to the oligodeoxynucleotide the SSOMV can contain a 5′ blocking substituent that is attached to the 5′ terminal carbons through a linker. The chemistry of the linker is not critical other than its length, which should preferably be at least 6 atoms long and that the linker should be flexible. A variety of non-toxic substituents such as biotin, cholesterol or other steroids or a non-intercalating cationic fluorescent dye can be used. Particularly preferred as reagents to make SSOMV are the reagents sold as Cy3™ and Cy5™ by Glen Research, Sterling Va. (now GE Healthcare), which are blocked phosphoroamidites that upon incorporation into an oligonucleotide yield 3,3,3′,3′-tetramethyl N,N′-isopropyl substituted indomonocarbocyanine and indodicarbocyanine dyes, respectively. Cy3 is the most preferred. When the indocarbocyanine is N-oxyalkyl substituted it can be conveniently linked to the 5′ terminal of the oligodeoxynucleotide through as a phosphodiester with a 5′ terminal phosphate. The chemistry of the dye linker between the dye and the oligodeoxynucleotide is not critical and is chosen for synthetic convenience. When the commercially available Cy3 phosphoramidite is used as directed the resulting 5′ modification consists of a blocking substituent and linker together which are a N-hydroxypropyl, N′-phosphatidylpropyl 3,3,3′,3′-tetramethyl indomonocarbocyanine.

In a preferred embodiment the indocarbocyanine dye is tetra substituted at the 3 and 3′ positions of the indole rings. Without limitation as to theory these substitutions prevent the dye from being an intercalating dye. The identity of the substituents at these positions is not critical. The SSOMV can in addition have a 3′ blocking substituent. Again the chemistry of the 3′ blocking substituent is not critical.

In another embodiment the oligonucleotide may be a single-stranded oligodeoxynucleotide having a 3′ end nucleotide, a 5′ end nucleotide, having at least 25 deoxynucleotides and not more than 65 deoxynucleotides, and having a sequence comprising at least two regions each of at least 8 deoxynucleotides that are each, respectively, identical to at least two regions of the targeted chromosomal gene, which regions together are at least 24 nucleotides in length, and which regions are separated by at least one nucleotide in the sequence of the targeted chromosomal gene or in the sequence of the oligodeoxynucleotide or both such that the sequence of the oligodeoxynucleotide is not identical to the sequence of the targeted chromosomal gene. See U.S. Pat. No. 6,271,360 which is incorporated herein by reference.

The mutations herein described might also be obtained by mutagenesis (random, somatic or directed) and other DNA editing or nucleases using a repair template including, but not limited to, gene targeting using zinc finger nucleases, using Transcription Activator-Like Effector Nucleases (TALENs), using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs). These nucleases can be plasmid (DNA) based, RNA and/or protein.

Microcarriers and Microfibers

The use of metallic microcarriers (microspheres) for introducing large fragments of DNA into plant cells having cellulose cell walls by projectile penetration is well known to those skilled in the relevant art (henceforth biolistic delivery). U.S. Pat. Nos. 4,945,050; 5,100,792 and 5,204,253 describe general techniques for selecting microcarriers and devices for projecting them. U.S. Pat. Nos. 5,484,956 and 5,489,520 describe the preparation of fertile transgenic corn using microprojectile bombardment of corn callus tissue. The biolistic techniques are also used in transforming immature corn embryos.

Specific conditions for using microcarriers in the methods of the present disclosure are described in International Publication WO 99/07865. In an illustrative technique, ice cold microcarriers (60 mg/ml), mixed duplex oligonucleotide (60 mg/ml) 2.5 M CaCl₂) and 0.1 M spermidine are added in that order; the mixture is gently agitated, e.g., by vortexing, for 10 minutes and let stand at room temperature for 10 minutes, whereupon the microcarriers are diluted in 5 volumes of ethanol, centrifuged and resuspended in 100% ethanol. Good results can be obtained with a concentration in the adhering solution of 8-10 μg/μl microcarriers, 14-17 μg/ml mixed duplex oligonucleotide, 1.1-1.4 M CaCl₂) and 18-22 mM spermidine. Optimal results were observed under the conditions of 8 μg/μl microcarriers, 16.5 μg/ml mixed duplex oligonucleotide, 1.3 M CaCl₂) and 21 mM spermidine.

Oligonucleobases can also be introduced into plant cells for the practice of the present disclosure using microfibers to penetrate the cell wall and cell membrane. U.S. Pat. No. 5,302,523 to Coffee, R., and Dunwell, J. M. (1994) describes the use of 30×0.5 μm and 10×0.3 μm silicon carbide fibers to facilitate transformation of suspension maize cultures of Black Mexican Sweet. Any mechanical technique that can be used to introduce DNA for transformation of a plant cell using microfibers can be used to deliver oligonucleobases for use in making the present SHP mutants. The process disclosed by Coffee, R., and Dunwell, J. M. (1994) in U.S. Pat. No. 5,302,523 can be employed with regenerable plant cell materials to introduce the present oligonucleobases to effect the mutation of the SHP gene.

An illustrative technique for microfiber delivery of an oligonucleobase is as follows: Sterile microfibers (2 μg) are suspended in 150 μl of plant culture medium containing about 10 μg of a mixed duplex oligonucleotide. A suspension culture is allowed to settle, and equal volumes of packed cells and the sterile fiber/nucleotide suspension are vortexed for 10 minutes and plated. Selective media are applied immediately or with a delay of up to about 120 hours as is appropriate for the particular trait.

Electroporation

In an alternative embodiment, the oligonucleobases can be delivered to the plant cell by electroporation of a protoplast derived from a plant part according to techniques that are well-known to one of ordinary skill in the art. See, e.g., Gallois et al., 1996, in Methods in Molecular Biology 55:89-107, Humana Press, Totowa, N.J.; Kipp et al., 1999, in Methods in Molecular Biology 133:213-221, Humana Press, Totowa, N.J.

Oligonucleobases can also be introduced into microspores by electroporation. Upon release of the tetrad, the microspore is uninucleate and thin-walled. It begins to enlarge and develops a germpore before the exine forms. A microspore at this stage is potentially more amenable to transformation with exogenous DNA than other plant cells. In addition, microspore development can be altered in vitro to produce either haploid embryos or embryogenic callus that can be regenerated into plants (Coumans et al., Plant Cell Rep. 7:618-621, 1989; Datta et al., Plant Sci. 67:83-88, 1990; Maheshwari et al., Am. J Bot. 69:865-879, 1982; Schaeffer, Adv. In Cell Culture 7:161-182, 1989; Swanson et al., Plant Cell Rep. 6:94-97, 1987). Thus, transformed microspores can be regenerated directly into haploid plants or dihaploid fertile plants upon chromosome doubling by standard methods. See also co-pending application U.S. Ser. No. 09/680,858 entitled Compositions and Methods for Plant Genetic Modification which is incorporated herein by reference.

Microspore electroporation can be practiced with any plant species for which microspore culture is possible, including but not limited to plants in the families Graminae, Leguminoceae, Cruciferaceae, Solanaceae, Cucurbitaceae, Rosaceae, Poaceae, Lilaceae, Rutaceae, Vitaceae, including such species as corn (Zea mays), wheat (Triticum aestivum), rice (Oryza saliva), oats, barley, canola (Brassica napus, Brassica rapa, Brassica oleracea, and Brassica juncea), cotton (Gossypium hirsuitum L.), various legume species (e.g., soybean (Glycine max), pea (Pisum sativum), etc.), grapes (Vitis vinifera), and a host of other important crop plants. Microspore embryogenesis, both from anther and microspore culture, has been described in more than 170 species, belonging to 68 genera and 28 families of dicotyledons and monocotyledons (Raghavan, Embryogenesis in Angiosperms: A Developmental and Experimental Study, Cambridge University Press, Cambridge, England, 1986; Rhagavan, Cell Differentiation 21:213-226, 1987; Raemakers et al., Euphytica 81:93-107, 1995). For a detailed discussion of microspore isolation, culture, and regeneration of double haploid plants from microspore-derived embryos (MDE) in Brassica napus L., see Nehlin, The Use of Rapeseed (Brassica napus L.) Microspores as a Tool for Biotechnological Applications, doctoral thesis, Swedish University of Agricultural Sciences, Uppsala, Sweden, 1999; also Nehlin et al., Plant Sci. 111:219-227, 1995, and Nehlin et al., Plant Sci. 111:219-227, 1995). Chromosome doubling from microspore or anther culture is a well-established technique for production of double-haploid homozygous plant lines in several crops (Heberle-Bors et al., In vitro pollen cultures: Progress and perspectives. In: Pollen Biotechnology. Gene expression and allergen characterization, vol. 85-109, ed. Mohapatra, S. S., and Knox, R. B., Chapman and Hall, New York, 1996).

Microspore electroporation methods are described in Jardinaud et al., Plant Sci. 93:177-184, 1993, and Fennell and Hauptman, Plant Cell Reports 11:567-570, 1992. Methods for electroporation of MDON into plant protoplasts can also be adapted for use in microspore electroporation.

Whiskers Technique

In yet another alternative embodiment, the oligonucleobase can be delivered to the plant cell by whiskers or microinjection of the plant cell. The so-called whiskers technique is performed essentially as described in Frame et al., 1994, Plant J. 6:941-948. The oligonucleobase is added to the whiskers and used to transform the plant cells. The oligonucleobase may be co-incubated with plasmids comprising sequences encoding proteins capable of forming recombinase complexes in plant cells such that recombination is catalyzed between the oligonucleotide and the target sequence in the SHP gene.

Other Delivery Methods

In an alternative embodiment, nucleic acids are embedded in microbeads composed of calcium alginate and taken up by plant protoplasts in the presence of the membrane-modifying agent polyethylene glycol (see, e.g., Sone et al., 2002; Liu et al., 2004).

In an alternative embodiment, nucleic acids frozen in water and introduced into plant cells by bombardment in the form of microparticles (see, e.g., Gilmore, 1991, U.S. Pat. No. 5,219,746; Brinegar et al.).

In an alternative embodiment, nucleic acids attached to nanoparticles are introduced into intact plant cells by incubation of the cells in a suspension containing the nanoparticle (see, e.g., Pasupathy et al., 2008) or by delivering them into intact cells through particle bombardment or into protoplasts by co-incubation (see, e.g., Torney et al., 2007).

In an alternative embodiment, nucleic acids complexed with penetrating peptides are delivered into cells by co-incubation (see, e.g., Chugh et al., 2008, WO 2008148223 A1; Eudes and Chugh).

In an alternative embodiment, nucleic acids are introduced into intact cells through electroporation (see, e.g., He et al., 1998, U.S. 2003/0115641 A1, Dobres et al.).

In an alternative embodiment, nucleic acids are delivered into cells of dry embryos by soaking them in a solution with nucleic acids (by soaking dry embryos in (see, e.g., Töpfer et al., 1989, Senaratna et al., 1991).

Targeted Gene Modification

Targeted genetic modification mediated by oligonucleotides is a valuable technique for use in the specific alteration of short stretches of DNA to create deletions, short insertions, and point mutations and may be used in conjunction with the disclosures herein, for example to cause one or more of the SHP mutations contemplated herein. These methods may in some embodiments involve DNA pairing/annealing, followed by a DNA repair event. First, the nucleic acid anneals with its complementary strand in the double-stranded DNA in a process mediated by cellular protein factors. This annealing creates a centrally located mismatched base pair (in the case of a point mutation), resulting in a structural perturbation that most likely stimulates the endogenous protein machinery to initiate the second step in the repair process: site-specific modification of the chromosomal sequence and/or that in organelles (e.g., mitochondria and chloroplasts). This newly introduced mismatch induces the DNA repair machinery to perform a second repair event, leading to the final revision of the target site. The present methods and compositions in various aspects and embodiments disclosed herein, may improve the methods by providing novel approaches which increase the availability of DNA repair components, thus increasing the efficiency and reproducibility of gene repair-mediated modifications to targeted nucleic acids.

Efficient methods for site-directed genomic modifications are desirable for research, clinical gene therapy, industrial microbiology and agriculture. One approach utilizes triplex-forming oligonucleotides (TFO) which bind as third strands to duplex DNA in a sequence-specific manner, to mediate directed mutagenesis. Such TFO can act either by delivering a tethered mutagen, such as psoralen or chlorambucil (Havre et al., Proc Nat'l Acad Sci, U.S.A. 90:7879-7883, 1993; Havre et al., J Virol 67:7323-7331, 1993; Wang et al., Mol Cell Biol 15:1759-1768, 1995; Takasugi et al., Proc Nat'l Acad Sci, U.S.A. 88:5602-5606, 1991; Belousov et al., Nucleic Acids Res 25:3440-3444, 1997), or by binding with sufficient affinity to provoke error-prone repair (Wang et al., Science 271:802-805, 1996).

Another strategy for genomic modification that may be used in conjunction with the compositions and methods herein involves the induction of homologous recombination between an exogenous DNA fragment and the targeted gene. This approach has been used successfully to target and disrupt selected genes in mammalian cells and has enabled the production of transgenic mice carrying specific gene knockouts (Capeechi et al., Science 244:1288-1292, 1989; Wagner, U.S. Pat. No. 4,873,191). This approach involves the transfer of selectable markers to allow isolation of the desired recombinants. Without selection, the ratio of homologous to non-homologous integration of transfected DNA in typical gene transfer experiments is low, usually in the range of 1:1000 or less (Sedivy et al., Gene Targeting, W. H. Freeman and Co., New York, 1992). This low efficiency of homologous integration limits the utility of gene transfer for experimental use or gene therapy. The frequency of targeted mutation can be enhanced by damage to the target site from UV irradiation and selected carcinogens (Wang et al., Mol Cell Biol 8:196-202, 1988) as well as by site-specific endonucleases (Sedivy et al, Gene Targeting, W. H. Freeman and Co., New York, 1992; Rouet et al., Proc Nat'l Acad Sci, U.S.A. 91:6064-6068, 1994; Segal et al., Proc Nat'l Acad Sci, U.S.A. 92:806-810, 1995). In addition, DNA damage induced by triplex-directed psoralen photoadducts can stimulate recombination within and between extrachromosomal vectors (Segal et al., Proc Nat'l Acad Sci, U.S.A. 92:806-810, 1995; Faruqi et al., Mol Cell Biol 16:6820-6828, 1996; Glazer, U.S. Pat. No. 5,962,426).

Linear donor fragments are more efficacious for targeted mutation than their circular counterparts (Folger et al., Mol Cell Biol 2:1372-1387, 1982). Recombination can in certain embodiments also be influenced by the length of uninterrupted homology between both the donor and target sites, with short fragments often appearing to be ineffective substrates (Rubnitz et al., Mol Cell Biol 4:2253-2258, 1984). Nonetheless, the use of short fragments of DNA or DNA/RNA hybrids for gene correction is the focus of various strategies. (Kunzelmann et al., Gene Ther 3:859-867, 1996).

“Nucleic acid sequence,” “nucleotide sequence” and “polynucleotide sequence” as used herein refer to an oligonucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded and represent the sense or antisense strand.

As used herein, the terms “oligonucleotide” and “oligomer” refer to a polymer of nucleobases. In some embodiments an “oligonucleotide” or “oligomer” may be of at least about 8 nucleobases or may have as many as about 1,500 nucleobases or more. In certain embodiments, an “oligonucleotide” or “oligomer” may be any length as contemplated herein.

The terms “DNA-modifying molecule” and “DNA-modifying reagent” as used herein refer to a molecule which is capable of recognizing and specifically binding to a nucleic acid sequence in the genome of a cell, and which is capable of modifying a target nucleotide sequence within the genome, wherein the recognition and specific binding of the DNA-modifying molecule to the nucleic acid sequence is protein-independent. The term “protein-independent” as used herein in connection with a DNA-modifying molecule means that the DNA-modifying molecule does not require the presence and/or activity of a protein and/or enzyme for the recognition of, and/or specific binding to, a nucleic acid sequence. DNA-modifying molecules are exemplified, but not limited to triplex forming oligonucleotides, peptide nucleic acids, polyamides, and oligonucleotides which are intended to promote gene conversion. The DNA-modifying molecules of the present disclosure are in certain embodiments distinguished from the prior art's nucleic acid sequences which are used for homologous recombination (Wong & Capecchi, Molec. Cell. Biol. 7:2294-2295, 1987) in that the prior art's nucleic acid sequences which are used for homologous recombination are protein-dependent. The term “protein-dependent” as used herein in connection with a molecule means that the molecule requires the presence and/or activity of a protein and/or enzyme for the recognition of, and/or specific binding of the molecule to, a nucleic acid sequence. Methods for determining whether a DNA-modifying molecule requires the presence and/or activity of a protein and/or enzyme for the recognition of, and/or specific binding to, a nucleic acid sequence are within the skill in the art (see, e.g., Dennis et al. Nucl. Acids Res. 27:4734-4742, 1999). For example, the DNA-modifying molecule may be incubated in vitro with the nucleic acid sequence in the absence of any proteins and/or enzymes. The detection of specific binding between the DNA-modifying molecule and the nucleic acid sequence demonstrates that the DNA-modifying molecule is protein-independent. On the other hand, the absence of specific binding between the DNA-modifying molecule and the nucleic acid sequence demonstrates that the DNA-modifying molecule is protein-dependent and/or requires additional factors.

“Triplex forming oligonucleotide” (TFO) is defined as a sequence of DNA or RNA that is capable of binding in the major grove of a duplex DNA or RNA helix to form a triple helix. Although the TFO is not limited to any particular length, a preferred length of the TFO is 250 nucleotides or less, 200 nucleotides or less, or 100 nucleotides or less, or from 5 to 50 nucleotides, or from 10 to 25 nucleotides, or from 15 to 25 nucleotides. Although a degree of sequence specificity between the TFO and the duplex DNA is necessary for formation of the triple helix, no particular degree of specificity is required, as long as the triple helix is capable of forming. Likewise, no specific degree of avidity or affinity between the TFO and the duplex helix is required as long as the triple helix is capable of forming. While not intending to limit the length of the nucleotide sequence to which the TFO specifically binds in one embodiment, the nucleotide sequence to which the TFO specifically binds is from 1 to 100, in some embodiments from 5 to 50, yet other embodiments from 10 to 25, and in other embodiments from 15 to 25, nucleotides. Additionally, “triple helix” is defined as a double-helical nucleic acid with an oligonucleotide bound to a target sequence within the double-helical nucleic acid. The “double-helical” nucleic acid can be any double-stranded nucleic acid including double-stranded DNA, double-stranded RNA and mixed duplexes of DNA and RNA. The double-stranded nucleic acid is not limited to any particular length. However, in preferred embodiments it has a length of greater than 500 bp, in some embodiments greater than 1 kb and in some embodiments greater than about 5 kb. In many applications the double-helical nucleic acid is cellular, genomic nucleic acid. The triplex forming oligonucleotide may bind to the target sequence in a parallel or anti-parallel manner.

“Peptide Nucleic Acids,” “polyamides” or “PNA” are nucleic acids wherein the phosphate backbone is replaced with an N-aminoethylglycine-based polyamide structure. PNAs have a higher affinity for complementary nucleic acids than their natural counter parts following the Watson-Crick base-pairing rules. PNAs can form highly stable triple helix structures with DNA of the following stoichiometry: (PNA)2.DNA. Although the peptide nucleic acids and polyamides are not limited to any particular length, a preferred length of the peptide nucleic acids and polyamides is 200 nucleotides or less, in some embodiments 100 nucleotides or less, and in some embodiments from 5 to 50 nucleotides long. While not intending to limit the length of the nucleotide sequence to which the peptide nucleic acid and polyamide specifically binds, in one embodiment, the nucleotide sequence to which the peptide nucleic acid and polyamide specifically bind is from 1 to 100, in some embodiments from 5 to 50, yet other embodiments from 5 to 25, and other embodiments from 5 to 20, nucleotides.

The term “capable of modifying DNA” or “DNA modifying means” refers to procedures, as well as endogenous or exogenous agents or reagents that can induce, or can aid in the induction of, changes to the nucleotide sequence of a targeted segment of DNA. Such changes may be made by the deletion, addition or substitution of one or more bases on the targeted DNA segment. It is not necessary that the DNA sequence changes confer functional changes to any gene encoded by the targeted sequence. Furthermore, it is not necessary that changes to the DNA be made to any particular portion or percentage of the cells.

The term “nucleotide sequence of interest” refers to any nucleotide sequence, the manipulation of which may be deemed desirable for any reason, by one of ordinary skill in the art. Such nucleotide sequences include, but are not limited to, coding sequences of structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.), and non-coding regulatory sequences that do not encode an mRNA or protein product (e.g., promoter sequence, enhancer sequence, polyadenylation sequence, termination sequence, regulatory RNAs such as miRNA, etc.).

“Amino acid sequence,” “polypeptide sequence,” “peptide sequence” and “peptide” are used interchangeably herein to refer to a sequence of amino acids.

“Target sequence,” as used herein, refers to a double-helical nucleic acid comprising a sequence that is the subject of interest. In some embodiments a target sequence may be greater than 8 nucleotides in length and in some embodiments less than 1,500 nucleotides in length. In some embodiments, the target sequence is between 8 to 30 bases. In some embodiments the target sequence may be between about 75 and 250 bases in length. In certain embodiments the target sequence may be a length complimentary to the length of an oligonucleotide as contemplated herein. The target sequence, in general, is defined by the nucleotide sequence on one of the strands on the double-helical nucleic acid.

As used herein, a “purine-rich sequence” or “polypurine sequence” when made in reference to a nucleotide sequence on one of the strands of a double-helical nucleic acid sequence is defined as a contiguous sequence of nucleotides wherein greater than 50% of the nucleotides of the target sequence contain a purine base. However, it is preferred that the purine-rich target sequence contain greater than 60% purine nucleotides, in some embodiments greater than 75% purine nucleotides, in other embodiments greater than 90% purine nucleotides and yet other embodiments 100% purine nucleotides.

As used herein, a “pyrimidine-rich sequence” or “polypyrimidine sequence” when made in reference to a nucleotide sequence on one of the strands of a double-helical nucleic acid sequence is defined as a contiguous sequence of nucleotides wherein greater that 50% of the nucleotides of the target sequence contain a pyrimidine base. However, it is preferred that the pyrimidine-rich target sequence contain greater than 60% pyrimidine nucleotides and, in some embodiments, greater than 75% pyrimidine nucleotides. In some embodiments, the sequence contains greater than 90% pyrimidine nucleotides and, in other embodiments, is 100% pyrimidine nucleotides.

A “variant” of a first nucleotide sequence is defined as a nucleotide sequence which differs from the first nucleotide sequence (e.g., by having one or more deletions, insertions, or substitutions that may be detected using hybridization assays or using DNA sequencing). Included within this definition is the detection of alterations or modifications to the genomic sequence of the first nucleotide sequence. For example, hybridization assays may be used to detect (1) alterations in the pattern of restriction enzyme fragments capable of hybridizing to the first nucleotide sequence when comprised in a genome (i.e., RFLP analysis), (2) the inability of a selected portion of the first nucleotide sequence to hybridize to a sample of genomic DNA which contains the first nucleotide sequence (e.g., using allele-specific oligonucleotide probes), (3) improper or unexpected hybridization, such as hybridization to a locus other than the normal chromosomal locus for the first nucleotide sequence (e.g., using fluorescent in situ hybridization (FISH) to metaphase chromosomes spreads, etc.). One example of a variant is a mutated wild type sequence.

The terms “nucleic acid” and “unmodified nucleic acid” as used herein refer to any one of the known four deoxyribonucleic acid bases (i.e., guanine, adenine, cytosine, and thymine). The term “modified nucleic acid” refers to a nucleic acid whose structure is altered relative to the structure of the unmodified nucleic acid. Illustrative of such modifications would be replacement covalent modifications of the bases, such as alkylation of amino and ring nitrogens as well as saturation of double bonds.

As used herein, the terms “mutation” and “modification” and grammatical equivalents thereof when used in reference to a nucleic acid sequence are used interchangeably to refer to a deletion, insertion, substitution, strand break, and/or introduction of an adduct. A “deletion” is defined as a change in a nucleic acid sequence in which one or more nucleotides is absent. An “insertion” or “addition” is that change in a nucleic acid sequence which has resulted in the addition of one or more nucleotides. A “substitution” results from the replacement of one or more nucleotides by a molecule which is a different molecule from the replaced one or more nucleotides. For example, a nucleic acid may be replaced by a different nucleic acid as exemplified by replacement of a thymine by a cytosine, adenine, guanine, or uridine. Pyrimidine to pyrimidine (e.g. C to T or T to C nucleotide substitutions) or purine to purine (e.g. G to A or A to G nucleotide substitutions) are termed transitions, whereas pyrimidine to purine or purine to pyrimidine (e.g. G to T or G to C or A to T or A to C) are termed transversions. Alternatively, a nucleic acid may be replaced by a modified nucleic acid as exemplified by replacement of a thymine by thymine glycol. Mutations may result in a mismatch. The term “mismatch” refers to a non-covalent interaction between two nucleic acids, each nucleic acid residing on a different polynucleic acid sequence, which does not follow the base-pairing rules. For example, for the partially complementary sequences 5′-AGT-3′ and 5′-AAT-3′, a G-A mismatch (a transition) is present. The terms “introduction of an adduct” or “adduct formation” refer to the covalent or non-covalent linkage of a molecule to one or more nucleotides in a DNA sequence such that the linkage results in a reduction (in some embodiments from 10% to 100%, in other embodiments from 50% to 100%, and in some embodiments from 75% to 100%) in the level of DNA replication and/or transcription.

The term “DNA cutter” refers to a moiety that effects a strand break. Non-limited examples include meganucleases, TALEs/TALENs, antibiotics, zinc fingers and CRISPRs or CRISPR/Cas systems.

The term “strand break” when made in reference to a double stranded nucleic acid sequence includes a single-strand break and/or a double-strand break. A single-strand break (a nick) refers to an interruption in one of the two strands of the double stranded nucleic acid sequence. This contrasts with a double-strand break which refers to an interruption in both strands of the double stranded nucleic acid sequence, which may result in blunt or staggered ends. Strand breaks may be introduced into a double stranded nucleic acid sequence either directly (e.g., by ionizing radiation or treatment with certain chemicals) or indirectly (e.g., by enzymatic incision at a nucleic acid base). In certain embodiments, a DNA cutter may have selectivity for certain specific sequences, such as in the case of a CRISPR, a zinc finger, a meganuclease, a TALEN as described herein.

The terms “mutant cell” and “modified cell” refer to a cell which contains at least one modification in the cell's genomic sequence.

The term “portion” when used in reference to a nucleotide sequence refers to fragments of that nucleotide sequence. The fragments may range in size from 5 nucleotide residues to the entire nucleotide sequence minus one nucleic acid residue.

DNA molecules are said to have “5′ ends” and “3′ ends” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5′ phosphate of one mononucleotide pentose ring is attached to the 3′ oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotide is referred to as the “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the “3′ end” if its 3′ oxygen is not linked to a 5′ phosphate of another mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5′ and 3′ ends. In either a linear or circular DNA molecule, discrete elements are referred to as being “upstream” or 5′ of the “downstream” or 3′ elements. This terminology reflects that transcription proceeds in a 5′ to 3′ direction along the DNA strand. The promoter and enhancer elements which direct transcription of a linked gene are generally located 5′ or upstream of the coding region. However, enhancer elements can exert their effect even when located 3′ of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3′ or downstream of the coding region.

The term “recombinant DNA molecule” as used herein refers to a DNA molecule which is comprised of segments of DNA joined together by means of molecular biological techniques.

The term “recombinant protein” or “recombinant polypeptide” as used herein refers to a protein molecule which is expressed using a recombinant DNA molecule.

As used herein, the terms “vector” and “vehicle” are used interchangeably in reference to nucleic acid molecules that transfer one or more DNA segment from one cell to another.

The terms “in operable combination,” “in operable order” and “operably linked” as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The terms also refer to the linkage of amino acid sequences in such a manner so that a functional protein is produced.

The term “transfection” as used herein refers to the introduction of foreign DNA into cells. Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofectin, protoplast fusion, retroviral infection, biolistics (i.e., particle bombardment) and the like.

As used herein, the terms “complementary” or “complementarity” are used in reference to “polynucleotides” and “oligonucleotides” (which are interchangeable terms that refer to a sequence of nucleotides) related by the base-pairing rules. For example, the sequence “5′-CAGT-3′,” is complementary to the sequence “5′-ACTG-3′.” Complementarity can be “partial” or “total”. “Partial” complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules. “Total” or “complete” complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules. The degree of complementarity between nucleic acid strands may have significant effects on the efficiency and strength of hybridization between nucleic acid strands. This may be of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids. For the sake of convenience, the terms “polynucleotides” and “oligonucleotides” include molecules which include nucleosides.

The terms “homology” and “homologous” as used herein in reference to nucleotide sequences refer to a degree of complementarity with other nucleotide sequences. There may be partial homology or complete homology (i.e., identity). When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term “substantially homologous” refers to any nucleic acid sequence (e.g., probe) which can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above. A nucleotide sequence which is partially complementary, i.e., “substantially homologous,” to a nucleic acid sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid sequence. The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence to a target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested using a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.

Low stringency conditions comprise conditions equivalent to binding or hybridization at 68° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/l NaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5×Denhardt's reagent (50×Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)) and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 2.0×SSPE, 0.1% SDS at room temperature when a probe of about 100 to about 1000 nucleotides in length is employed.

In addition, conditions which promote hybridization under conditions of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.) are well known in the art. High stringency conditions, when used in reference to nucleic acid hybridization, comprise conditions equivalent to binding or hybridization at 68° C. in a solution consisting of 5×SSPE, 1% SDS, 5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1×SSPE and 0.1% SDS at 68° C. when a probe of about 100 to about 1000 nucleotides in length is employed.

It is well known in the art that numerous equivalent conditions may be employed to comprise low stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol), as well as components of the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions.

The term “equivalent” when made in reference to a hybridization condition as it relates to a hybridization condition of interest means that the hybridization condition and the hybridization condition of interest result in hybridization of nucleic acid sequences which have the same range of percent (%) homology. For example, if a hybridization condition of interest results in hybridization of a first nucleic acid sequence with other nucleic acid sequences that have from 50% to 70% homology to the first nucleic acid sequence, then another hybridization condition is said to be equivalent to the hybridization condition of interest if this other hybridization condition also results in hybridization of the first nucleic acid sequence with the other nucleic acid sequences that have from 50% to 70% homology to the first nucleic acid sequence.

As used herein, the term “hybridization” is used in reference to the pairing of complementary nucleic acids using any process by which a strand of nucleic acid joins with a complementary strand through base pairing to form a hybridization complex. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acids.

As used herein the term “hybridization complex” refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized to a solid support (e.g., a nylon membrane or a nitrocellulose filter as employed in Southern and Northern blotting, dot blotting or a glass slide as employed in in situ hybridization, including FISH (fluorescent in situ hybridization)).

As used herein, the term “Tm” is used in reference to the “melting temperature.” The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the Tm of nucleic acids is well known in the art. As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization, 1985). Other references include more sophisticated computations which take structural as well as sequence characteristics into account for the calculation of Tm.

As used herein the term “stringency” is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. “Stringency” typically occurs in a range from about Tm-5° C. (5° C. below the melting temperature of the probe) to about 20° C. to 25° C. below Tm. As will be understood by those of skill in the art, a stringent hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences.

The terms “specific binding,” “binding specificity,” and grammatical equivalents thereof when made in reference to the binding of a first nucleotide sequence to a second nucleotide sequence, refer to the preferential interaction between the first nucleotide sequence with the second nucleotide sequence as compared to the interaction between the second nucleotide sequence with a third nucleotide sequence. Specific binding is a relative term that does not require absolute specificity of binding; in other words, the term “specific binding” does not require that the second nucleotide sequence interact with the first nucleotide sequence in the absence of an interaction between the second nucleotide sequence and the third nucleotide sequence. Rather, it is sufficient that the level of interaction between the first nucleotide sequence and the second nucleotide sequence is greater than the level of interaction between the second nucleotide sequence with the third nucleotide sequence. “Specific binding” of a first nucleotide sequence with a second nucleotide sequence also means that the interaction between the first nucleotide sequence and the second nucleotide sequence is dependent upon the presence of a particular structure on or within the first nucleotide sequence; in other words the second nucleotide sequence is recognizing and binding to a specific structure on or within the first nucleotide sequence rather than to nucleic acids or to nucleotide sequences in general. For example, if a second nucleotide sequence is specific for structure “A” that is on or within a first nucleotide sequence, the presence of a third nucleic acid sequence containing structure A will reduce the amount of the second nucleotide sequence which is bound to the first nucleotide sequence.

As used herein, the term “amplifiable nucleic acid” is used in reference to nucleic acids which may be amplified by any amplification method. It is contemplated that “amplifiable nucleic acid” will usually comprise “sample template.”

The terms “heterologous nucleic acid sequence” or “heterologous DNA” are used interchangeably to refer to a nucleotide sequence which is ligated to a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Heterologous DNA is not endogenous to the cell into which it is introduced but has been obtained from another cell. Generally, although not necessarily, such heterologous DNA encodes RNA and proteins that are not normally produced by the cell into which it is expressed. Examples of heterologous DNA include reporter genes, transcriptional and translational regulatory sequences, selectable marker proteins (e.g., proteins which confer drug resistance), etc.

“Amplification” is defined as the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction technologies well known in the art (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.). As used herein, the term “polymerase chain reaction” (“PCR”) refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195, and 4,683,202, hereby incorporated by reference, which describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. The length of the amplified segment of the desired target sequence is determined by the relative positions of two oligonucleotide primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the “polymerase chain reaction” (“PCR”). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified.”

With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of ³²P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.

One such preferred method, particularly for commercial applications, is based on the widely used TaqMan® real-time PCR technology and combines Allele-Specific PCR with a Blocking reagent (ASB-PCR) to suppress amplification of the wildtype allele. ASB-PCR can be used for detection of germ line or somatic mutations in either DNA or RNA extracted from any type of tissue, including formalin-fixed paraffin-embedded tumor specimens. A set of reagent design rules are developed enabling sensitive and selective detection of single point substitutions, insertions, or deletions against a background of wild-type allele in thousand-fold or greater excess. (Morlan J, Baker J, Sinicropi D Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method. PLoS ONE 4(2): e4584, 2009)

The terms “reverse transcription polymerase chain reaction” and “RT-PCR” refer to a method for reverse transcription of an RNA sequence to generate a mixture of cDNA sequences, followed by increasing the concentration of a desired segment of the transcribed cDNA sequences in the mixture without cloning or purification. Typically, RNA is reverse transcribed using a single primer (e.g., an oligo-dT primer) prior to PCR amplification of the desired segment of the transcribed DNA using two primers.

As used herein, the term “primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and of an inducing agent such as DNA polymerase and at a suitable temperature and pH). In some embodiments, the primer is single stranded for maximum efficiency in amplification but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. In some embodiments, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present disclosure will be labeled with any “reporter molecule,” so that it is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present disclosure be limited to any particular detection system or label.

As used herein, the terms “restriction endonucleases” and “restriction enzymes” refer to bacterial enzymes, each of which cut or nick double- or single-stranded DNA at or near a specific nucleotide sequence, for example, an endonuclease domain of a type IS restriction endonuclease (e.g., FokI can be used, as taught by Kim et al., 1996, Proc. Nat'l. Acad. Sci. USA, 6:1 156-60).

As used herein, the term “an oligonucleotide having a nucleotide sequence encoding a gene” means a nucleic acid sequence comprising the coding region of a gene, i.e. the nucleic acid sequence which encodes a gene product. The coding region may be present in either a cDNA, genomic DNA or RNA form. When present in a DNA form, the oligonucleotide may be single-stranded (i.e., the sense strand) or double-stranded. Additionally, “an oligonucleotide having a nucleotide sequence encoding a gene” may include suitable control elements such as enhancers, promoters, splice junctions, polyadenylation signals, etc. if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript. Further still, the coding region of the present disclosure may contain endogenous enhancers, splice junctions, intervening sequences, polyadenylation signals, etc.

Transcriptional control signals in eukaryotes comprise “enhancer” elements. Enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis, T. et al., Science 236:1237, 1987). Enhancer elements have been isolated from a variety of eukaryotic sources including genes in plant, yeast, insect and mammalian cells and viruses. The selection of a particular enhancer depends on what cell type is to be used to express the protein of interest.

The presence of “splicing signals” on an expression vector often results in higher levels of expression of the recombinant transcript. Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York, pp. 16.7-16.8, 1989). A commonly used splice donor and acceptor site is the splice junction from the 16S RNA of SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cells requires expression of signals directing the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length. The term “poly A site” or “poly A sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and are rapidly degraded. The poly A signal utilized in an expression vector may be “heterologous” or “endogenous.” An endogenous poly A signal is one that is found naturally at the 3′ end of the coding region of a given gene in the genome. A heterologous poly A signal is one which is isolated from one gene and placed 3′ of another gene.

The term “promoter,” “promoter element” or “promoter sequence” as used herein, refers to a DNA sequence which when placed at the 5′ end of (i.e., precedes) an oligonucleotide sequence is capable of controlling the transcription of the oligonucleotide sequence into mRNA. A promoter is typically located 5′ (i.e., upstream) of an oligonucleotide sequence whose transcription into mRNA it controls and provides a site for specific binding by RNA polymerase and for initiation of transcription.

The term “promoter activity” when made in reference to a nucleic acid sequence refers to the ability of the nucleic acid sequence to initiate transcription of an oligonucleotide sequence into mRNA.

The term “tissue specific” as it applies to a promoter refers to a promoter that can direct selective expression of an oligonucleotide sequence to a specific type of tissue in the relative absence of expression of the same oligonucleotide in a different type of tissue. Tissue specificity of a promoter may be evaluated by, for example, operably linking a reporter gene to the promoter sequence to generate a reporter construct, introducing the reporter construct into the genome of a plant or an animal such that the reporter construct is integrated into every tissue of the resulting transgenic animal, and detecting the expression of the reporter gene (e.g., detecting mRNA, protein, or the activity of a protein encoded by the reporter gene) in different tissues of the transgenic plant or animal. Selectivity need not be absolute. The detection of a greater level of expression of the reporter gene in one or more tissues relative to the level of expression of the reporter gene in other tissues shows that the promoter is specific for the tissues in which greater levels of expression are detected.

The term “cell type specific” as applied to a promoter refers to a promoter which is capable of directing selective expression of an oligonucleotide sequence in a specific type of cell in the relative absence of expression of the same oligonucleotide sequence in a different type of cell within the same tissue. The term “cell type specific” when applied to a promoter also means a promoter capable of promoting selective expression of an oligonucleotide in a region within a single tissue. Again, selectivity need not be absolute. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining as described herein. Briefly, tissue sections are embedded in paraffin, and paraffin sections are reacted with a primary antibody which is specific for the polypeptide product encoded by the oligonucleotide sequence whose expression is controlled by the promoter. As an alternative to paraffin sectioning, samples may be cryosectioned. For example, sections may be frozen prior to and during sectioning thus avoiding potential interference by residual paraffin. A labeled (e.g., peroxidase conjugated) secondary antibody which is specific for the primary antibody is allowed to bind to the sectioned tissue and specific binding detected (e.g., with avidin/biotin) by microscopy.

The terms “selective expression,” “selectively express” and grammatical equivalents thereof refer to a comparison of relative levels of expression in two or more regions of interest. For example, “selective expression” when used in connection with tissues refers to a substantially greater level of expression of a gene of interest in a particular tissue, or to a substantially greater number of cells which express the gene within that tissue, as compared, respectively, to the level of expression of, and the number of cells expressing, the same gene in another tissue (i.e., selectivity need not be absolute). Selective expression does not require, although it may include, expression of a gene of interest in a particular tissue and a total absence of expression of the same gene in another tissue. Similarly, “selective expression” as used herein in reference to cell types refers to a substantially greater level of expression of, or a substantially greater number of cells which express, a gene of interest in a particular cell type, when compared, respectively, to the expression levels of the gene and to the number of cells expressing the gene in another cell type.

The term “contiguous” when used in reference to two or more nucleotide sequences means the nucleotide sequences are ligated in tandem either in the absence of intervening sequences, or in the presence of intervening sequences which do not comprise one or more control elements.

As used herein, the terms “nucleic acid molecule encoding,” “nucleotide encoding,” “DNA sequence encoding” and “DNA encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.

The term “isolated” when used in relation to a nucleic acid, as in “an isolated oligonucleotide” refers to a nucleic acid sequence that is separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is nucleic acid present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids are nucleic acids such as DNA and RNA which are found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs which encode a multitude of proteins. However, isolated nucleic acid encoding a polypeptide of interest includes, by way of example, such nucleic acid in cells ordinarily expressing the polypeptide of interest where the nucleic acid is in a chromosomal or extrachromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid or oligonucleotide may be present in single-stranded or double-stranded form. Isolated nucleic acid can be readily identified (if desired) by a variety of techniques (e.g., hybridization, dot blotting, etc.). When an isolated nucleic acid or oligonucleotide is to be utilized to express a protein, the oligonucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide may be single-stranded). Alternatively, it may contain both the sense and anti-sense strands (i.e., the oligonucleotide may be double-stranded).

As used herein, the term “purified” or “to purify” refers to the removal of one or more (undesired) components from a sample. For example, where recombinant polypeptides are expressed in bacterial host cells, the polypeptides are purified by the removal of host cell proteins thereby increasing the percent of recombinant polypeptides in the sample.

As used herein, the term “substantially purified” refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, in some embodiments 75% free and other embodiments 90% free from other components with which they are naturally associated. An “isolated polynucleotide” is, therefore, a substantially purified polynucleotide.

As used herein the term “coding region” when used in reference to a structural gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of a mRNA molecule. The coding region is bounded, in eukaryotes, on the 5′ side generally by the nucleotide triplet “ATG” which encodes the initiator methionine and on the 3′ side by one of the three triplets which specify stop codons (i.e., TAA, TAG, TGA).

By “coding sequence” is meant a sequence of a nucleic acid or its complement, or a part thereof, that can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof. Coding sequences include exons in a genomic DNA or immature primary RNA transcripts, which are joined together by the cell's biochemical machinery to provide a mature mRNA. The anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

By “non-coding sequence” is meant a sequence of a nucleic acid or its complement, or a part thereof that is not transcribed into amino acid in vivo, or where tRNA does not interact to place or attempt to place an amino acid. Non-coding sequences include both intron sequences in genomic DNA or immature primary RNA transcripts, and gene-associated sequences such as promoters, enhancers, silencers, etc.

As used herein, the term “structural gene” or “structural nucleotide sequence” refers to a DNA sequence coding for RNA or a protein which does not control the expression of other genes. In contrast, a “regulatory gene” or “regulatory sequence” is a structural gene which encodes products (e.g., transcription factors) which control the expression of other genes.

As used herein, the term “regulatory element” refers to a genetic element which controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements include splicing signals, polyadenylation signals, termination signals, etc.

As used herein, the term “peptide transcription factor binding site” or “transcription factor binding site” refers to a nucleotide sequence which binds protein transcription factors and, thereby, controls some aspect of the expression of nucleic acid sequences. For example, Sp-1 and API (activator protein 1) binding sites are examples of peptide transcription factor binding sites.

As used herein, the term “gene” means the deoxyribonucleotide sequences comprising the coding region of a structural gene. A “gene” may also include non-translated sequences located adjacent to the coding region on both the 5′ and 3′ ends such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5′ of the coding region and which are present on the mRNA are referred to as 5′ non-translated sequences. The sequences which are located 3′ or downstream of the coding region and which are present on the mRNA are referred to as 3′ non-translated sequences. The term “gene” encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.” Introns are segments of a gene which are transcribed into heterogenous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5′ and 3′ end of the sequences which are present on the RNA transcript. These sequences are referred to as “flanking” sequences or regions (these flanking sequences are located 5′ or 3′ to the non-translated sequences present on the mRNA transcript). The 5′ flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene. The 3′ flanking region may contain sequences which direct the termination of transcription, post-transcriptional cleavage and polyadenylation.

A “non-human animal” refers to any animal which is not a human and includes vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc. Preferred non-human animals are selected from the order Rodentia. “Non-human animal” additionally refers to amphibians (e.g. Xenopus), reptiles, insects (e.g. Drosophila) and other non-mammalian animal species.

As used herein, the term “transgenic” refers to an organism or cell that has DNA derived from another organism inserted into which becomes integrated into the genome either of somatic and/or germ line cells of the plant or animal. A “transgene” means a DNA sequence which is partly or entirely heterologous (i.e., not present in nature) to the plant or animal in which it is found, or which is homologous to an endogenous sequence (i.e., a sequence that is found in the animal in nature) and is inserted into the plant' or animal's genome at a location which differs from that of the naturally occurring sequence. Transgenic plants or animals which include one or more transgenes are within the scope of this disclosure. Additionally, a “transgenic” as used herein refers to an organism that has had one or more genes modified and/or “knocked out” (made non-functional or made to function at reduced level, e.g., a “knockout” mutation) by the disclosure's methods, by homologous recombination, TFO mutation or by similar processes. For example, in some embodiments, a transgenic organism or cell includes inserted DNA that includes a foreign promoter and/or coding region.

A “transformed cell” is a cell or cell line that has acquired the ability to grow in cell culture for multiple generations, the ability to grow in soft agar, and/or the ability to not have cell growth inhibited by cell-to-cell contact. In this regard, transformation refers to the introduction of foreign genetic material into a cell or organism. Transformation may be accomplished by any method known which permits the successful introduction of nucleic acids into cells and which results in the expression of the introduced nucleic acid. “Transformation” includes but is not limited to such methods as transfection, microinjection, electroporation, nucleofection and lipofection (liposome-mediated gene transfer). Transformation may be accomplished through use of any expression vector. For example, the use of baculovirus to introduce foreign nucleic acid into insect cells is contemplated. The term “transformation” also includes methods such as P-element mediated germline transformation of whole insects. Additionally, transformation refers to cells that have been transformed naturally, usually through genetic mutation.

As used herein “exogenous” means that the gene encoding the protein is not normally expressed in the cell. Additionally, “exogenous” refers to a gene transfected into a cell to augment the normal (i.e. natural) level of expression of that gene.

A peptide sequence and nucleotide sequence may be “endogenous” or “heterologous” (i.e., “foreign”). The term “endogenous” refers to a sequence which is naturally found in the cell into which it is introduced so long as it does not contain some modification relative to the naturally-occurring sequence. The term “heterologous” refers to a sequence which is not endogenous to the cell into which it is introduced. For example, heterologous DNA includes a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Heterologous DNA also includes a nucleotide sequence which is naturally found in the cell into which it is introduced, and which contains some modification relative to the naturally-occurring sequence. Generally, although not necessarily, heterologous DNA encodes heterologous RNA and heterologous proteins that are not normally produced by the cell into which it is introduced. Examples of heterologous DNA include reporter genes, transcriptional and translational regulatory sequences, DNA sequences which encode selectable marker proteins (e.g., proteins which confer drug resistance), etc.

In certain aspects and embodiments of the disclosures herein, provided are methods for introducing a gene repair oligonucleobase (GRON)-mediated mutation into a target deoxyribonucleic acid (DNA) sequence in a plant cell; for example, for the purpose of modifying an SHP gene such as provided herein. In certain embodiments the methods may include, inter alia, culturing the plant cell under conditions that increase one or more cellular DNA repair processes prior to, and/or coincident with, delivery of a GRON into the plant cell; and/or delivery of a GRON into the plant cell greater than 15 bases in length, the GRON optionally comprising one or more; or two or more; mutation sites (such as SHP mutation sites as provided herein) for introduction into the target DNA.

A “gene repair oligonucleotide” or “GRON” as used herein means an oligonucleobase (e.g., mixed duplex oligonucleotides, non-nucleotide containing molecules, single stranded oligodeoxynucleotides, double stranded oligodeoxynucleotides and other gene repair molecules) that can under certain conditions direct single, or in some embodiments multiple, nucleotide deletions, insertions or substitutions in a DNA sequence. This oligonucleotide-mediated gene repair editing of the genome may comprise both non-homology-based repair systems (e.g., non-homologous end joining) and homology-based repair systems (e.g., homology-directed repair). The GRON is typically designed to align in register with a genomic target except for the designed mismatch(es). These mismatches can be recognized and corrected by harnessing one or more of the cell's endogenous DNA repair systems. In some embodiments a GRON or oligonucleotide can be designed to contain multiple differences when compared to the organism's target sequence. These differences may not all affect the protein sequence translated from said target sequence and in one or more cases be known as silent changes. Numerous variations of GRON structure, chemistry and function are described elsewhere herein. In various embodiments, a GRON as used herein may have one or more modifications. For example, a GRON as used herein may have one or more modifications that attract DNA repair machinery to the targeted (mismatch) site and/or that prevent recombination of part or all of the GRON (other than the desired targeted deletions, insertions, substitutions or the like) into the genomic DNA of the target DNA sequence and/or that increase the stability of the GRON.

In various embodiments, a GRON may have both RNA and DNA nucleotides and/or other types of nucleobases. In some embodiments, one or more of the DNA or RNA nucleotides comprise a modification.

In one aspect, provided is a method of causing a genetic change in a plant cell (for example a genetic change in a SHP gene), wherein the method involves exposing the cell to a DNA cutter and a GRON, for example a GRON that is modified as contemplated herein. In some embodiments the GRON may be modified such as with a Cy3 group, 3PS group, a 2′O-methyl group or other modification such as contemplated herein. In another aspect, provided is a plant cell that includes a DNA cutter and a GRON (such as a GRON that binds and/or modifies a SHP gene), for example where the GRON is modified such as with a Cy3 group, 3PS group, a 2′O-methyl group or other modification. In some embodiments the DNA cutter is one or more selected from a CRISPR which includes but is not limited to Cas9, Cpf1 and their corresponding homologues, orthologues and/or paralogues, a base editor, a TALEN, a zinc finger, meganuclease, and a DNA-cutting antibiotic. In some embodiments, the DNA cutter is a CRISPR. In some embodiments, the DNA cutter is a TALEN. The DNA cutter can be plasmid (DNA) based, RNA and/or protein. In some embodiments, the GRON is between 15 and 60 nucleobases in length; or between 30 and 40 nucleobases in length; or between 35 and 45 nucleobases in length; or between 20 and 70 nucleobases in length; or between 20 and 200 nucleobases in length; or between 30 and 180 nucleobases in length; or between 50 and 160 nucleobases in length; or between 70 and 150 nucleobases in length; or between 80 and 120 nucleobases in length; or between 90 and 110 nucleobases in length; or between 95 and 105 nucleobases in length; or between 80 and 300 nucleobases in length; or between 90 and 250 nucleobases in length; or between 100 and 150 nucleobases in length; or between 100 and 300 nucleobases in length; or between 150 and 200 nucleobases in length; or between 200 and 300 nucleobases in length; or between 250 and 350 nucleobases in length; or between 50 and 110 nucleobases in length; or between 50 and 200 nucleobases in length; or between 150 and 210 nucleobases in length; or between 20 and 1000 nucleobases in length; or between 100 and 1000 nucleobases in length; or between 200 and 1000 nucleobases in length; or between 300 and 1000 nucleobases in length; or between 400 and 1000 nucleobases in length; or between 500 and 1000 nucleobases in length; or between 600 and 1000 nucleobases in length; or between 700 and 1000 nucleobases in length; or between 800 and 1000 nucleobases in length; or between 900 and 1000 nucleobases in length; or between 300 and 800 nucleobases in length; or between 400 and 600 nucleobases in length; or between 500 and 700 nucleobases in length; or between 600 and 800 nucleobases in length; or longer than 30 nucleobases in length; or longer than 35 nucleobases in length; or longer than 40 nucleobases in length; or longer than 50 nucleobases in length; or longer than 60 nucleobases in length; or longer than 65 nucleobases in length; or longer than 70 nucleobases in length; or longer than 75 nucleobases in length; or longer than 80 nucleobases in length; or longer than 85 nucleobases in length; or longer than 90 nucleobases in length; or longer than 95 nucleobases in length; or longer than 100 nucleobases in length; or longer than 110 nucleobases in length; or longer than 125 nucleobases in length; or longer than 150 nucleobases in length; or longer than 165 nucleobases in length; or longer than 175 nucleobases in length; or longer than 200 nucleobases in length; or longer than 250 nucleobases in length; or longer than 300 nucleobases in length; or longer than 350 nucleobases in length; or longer than 400 nucleobases in length; or longer than 450 nucleobases in length; or longer than 500 nucleobases in length; or longer than 550 nucleobases in length; or longer than 600 nucleobases in length; or longer than 700 nucleobases in length; or longer than 800 nucleobases in length; or longer than 900 nucleobases in length.

GRONs may be targeted at both non-coding (NC) and coding (C) regions of a target gene.

The term “CRISPR” as used herein refers to elements; i.e., a cas (CRISPR associated) gene, transcript (e.g., mRNA) and/or protein and at least one CRISPR spacer sequence (Clustered Regularly Interspaced Short Palindromic Repeats, also known as SPIDRs—SPacer Interspersed Direct Repeats); that when effectively present or expressed in a cell could effect cleavage of a target DNA sequence via CRISPR/CAS cellular machinery such as described in e.g., Cong, L. et al., Science, vol. 339 no 6121 pp. 819-823 (2013); Jinek et al., Science, vol. 337:816-821 (2013); Wang et al., RNA, vol. 14, pp. 903-913 (2008); Zhang et al., Plant Physiology, vol. 161, pp. 20-27 (2013), Zhang et al, PCT Application No. PCT/US2013/074743; and Charpentier et al., PCT Application No. PCT/US2013/032589. In some embodiments, such as for example a CRISPR for use in a eukaryotic cell, a CRISPR as contemplated herein may also include an additional element that includes a sequence for one or more functional nuclear localization signals. CRISPRs as contemplated herein can be expressed in, administered to and/or present in a cell (such as a plant cell) in any of many ways or manifestations. For example, a CRISPR as contemplated herein may include or involve one or more of a CRISPR on a plasmid, a CRISPR nickase on a plasmid, a CRISPRa on a plasmid, or a CRISPRi on a plasmid as follows:

CRISPR on a plasmid: A recombinant expression vector comprising:

(i) a nucleotide sequence encoding a DNA-targeting RNA (e.g., guide RNA), wherein the DNA-targeting RNA comprises:

-   -   a. a first segment comprising a nucleotide sequence that is         complementary to a sequence in a target DNA (e.g., protospacer,         spacer, or crRNA); and     -   b. a second segment that interacts with a site-directed         modifying polypeptide (e.g., trans-activating crRNA or         tracrRNA); and

(ii) a nucleotide sequence encoding the site-directed modifying polypeptide (e.g., cas gene), wherein the site-directed polypeptide comprises:

-   -   a. an RNA-binding portion that interacts with the DNA-targeting         RNA (e.g., REC lobe); and     -   b. an activity portion that causes double-stranded breaks within         the target DNA (e.g., NUC lobe), wherein the site of the         double-stranded breaks within the target DNA is determined by         the DNA-targeting RNA.

CRISPR nickase on a plasmid. A recombinant expression vector comprising:

(i) a nucleotide sequence encoding a DNA-targeting RNA (e.g., guide RNA), wherein the DNA-targeting RNA comprises:

-   -   a. a first segment comprising a nucleotide sequence that is         complementary to a sequence in a target DNA (e.g., protospacer,         spacer, or crRNA); and     -   b. a second segment that interacts with a site-directed         modifying polypeptide (e.g., trans-activating crRNA or         tracrRNA); and

(ii) a nucleotide sequence encoding the site-directed modifying polypeptide (e.g., cas gene), wherein the site-directed polypeptide comprises:

-   -   a. an RNA-binding portion that interacts with the DNA-targeting         RNA (e.g., REC lobe); and     -   b. an activity portion that causes single-stranded breaks within         the target DNA (e.g., NUC lobe), wherein the site of the         single-stranded breaks within the target DNA is determined by         the DNA-targeting RNA.

CRISPRa on a plasmid. A recombinant expression vector comprising:

(i) a nucleotide sequence encoding a DNA-targeting RNA (e.g., guide RNA), wherein the DNA-targeting RNA comprises:

-   -   a. a first segment comprising a nucleotide sequence that is         complementary to a sequence in a target DNA (e.g., protospacer,         spacer, or crRNA); and     -   b. a second segment that interacts with a site-directed         modifying polypeptide (e.g., trans-activating crRNA or         tracrRNA); and

(ii) a nucleotide sequence encoding the site-directed modifying polypeptide (e.g., cas gene), wherein the site-directed polypeptide comprises:

-   -   a. an RNA-binding portion that interacts with the DNA-targeting         RNA (e.g., REC lobe); and     -   b. an activity portion that modulates transcription (e.g., NUC         lobe; in certain embodiments increases transcription) within the         target DNA, wherein the site of the transcriptional modulation         within the target DNA is determined by the DNA-targeting RNA.

CRISPRi on a plasmid. A recombinant expression vector comprising:

(i) a nucleotide sequence encoding a DNA-targeting RNA (e.g., guide RNA), wherein the DNA-targeting RNA comprises:

-   -   a. a first segment comprising a nucleotide sequence that is         complementary to a sequence in a target DNA (e.g., protospacer,         spacer, or crRNA); and     -   b. a second segment that interacts with a site-directed         modifying polypeptide (e.g., trans-activating crRNA or         tracrRNA); and

(ii) a nucleotide sequence encoding the site-directed modifying polypeptide (e.g., cas gene), wherein the site-directed polypeptide comprises:

-   -   a. an RNA-binding portion that interacts with the DNA-targeting         RNA (e.g., REC lobe); and     -   b. an activity portion that modulates transcription/translation         (e.g., NUC lobe; in some embodiments decreases         transcription/translation) within the target DNA, wherein the         site of transcriptional/translational modulation within the         target DNA is determined by the DNA-targeting RNA.

Each of the CRISPR on a plasmid, CRISPR nickase on a plasmid, CRISPRa on a plasmid, and CRISPRi on a plasmid may in some embodiments alternatively have one or more appropriate elements be administered, expressed or present in a cell as an RNA (e.g., mRNA) or a protein rather than on a plasmid. Delivery of protected mRNA may be as described in Kariko, et al, U.S. Pat. No. 8,278,036.

In some embodiments, each of the CRISPRi and CRISPRa may include a deactivated cas9 (dCas9). A deactivated cas9 still binds to target DNA, but does not have cutting activity. Nuclease-deficient Cas9 can result from D10A and H840A point mutations which inactivates its two catalytic domains.

In some embodiments, a CRISPRi inhibits transcription initiation or elongation via steric hindrance of RNA Polymerase II. CRISPRi can optionally be enhanced (CRISPRei) by fusion of a strong repressor domain to the C-terminal end of a dCas9 protein. In some embodiments, a repressor domain recruits and employs chromatin modifiers. In some embodiments, the repressor domain may include, but is not limited to domains as described in Kagale, S. et al., Epigenetics, vol. 6 no 2 pp 141-146 (2011):

(LxLxPP motif) (SEQ ID NO: 17) 1. LDLNRPPPVEN - OsERF3 repressor domain  (R/KLFGV motif) (SEQ ID NO: 18) 2. LRLFGVNM - AtBRD repressor domain   (R/KLFGV motif) (SEQ ID NO: 19) 3. LKLFGVWL - AtHsfB1 repressor domain   (EAR motif)  (SEQ ID NO: 20) 4. LDLELRLGFA - AtSUP repressor domain  (EAR motif) (SEQ ID NO: 21) 5.ERSNSIELRNSFYGRARTSPWSYGDYDNCQQDHDYLLGF SWPPRSYTCSFCKREFRSAQALGGHMNVHRRDRARLRLQQS PSSSSTPSPPYPNPNYSYSTMANSPPPHHSPLTLFPTLSPP SSPRYRAGLIRSLSPKSKHTPENACKTKKSSLLVEAGEATR FTSKDACKILRNDEIISLELEIGLINESEQDLDLELRLGFA*-  full AtSUP gene containing repressor domain  

In some embodiments, a CRISPRa activation of transcription achieved by use of dCas9 protein containing a fused C-terminal end transcriptional activator. In some embodiments, an activation may include, but is not limited to VP64 (4X VP16), AtERF98 activation domain, or AtERF98x4 concatemers such as described in Cheng, A W et al., Cell Research, pp 1-9 (2013); Perez-Pinera, P. et al., Nature Methods, vol. 10 pp 913-976 (2013); Maeder, M L. et al., Nature Methods, vol. 10 pp 977-979 (2013) and Mali, P., et al., Nature Biotech., vol. 31 pp 833-838 (2013).

In some embodiments the CRISPR includes a nickase. In certain embodiments, two or more CRISPR nickases are used. In some embodiments, the two or more nickases cut on opposite strands of target nucleic acid. In other embodiments, the two or more nickases cut on the same strand of target nucleic acid.

As used herein, “repressor protein” or “repressor” refers to a protein that binds to operator of DNA or to RNA to prevent transcription or translation, respectively.

As used herein, “repression” refers to inhibition of transcription or translation by binding of repressor protein to specific site on DNA or mRNA. In some embodiments, repression includes a significant change in transcription or translation level of at least 1.5 fold, in other embodiments at least two fold, and in other embodiments at least five fold.

As used herein, an “activator protein” or “activator” with regard to gene transcription and/or translation, refers to a protein that binds to operator of DNA or to RNA to enhance or increase transcription or translation, respectively.

As used herein with regard to gene transcription and/or translation, “activation” with regard to gene transcription and/or translation, refers to enhancing or increasing transcription or translation by binding of activator protein to specific site on DNA or mRNA. In some embodiments, activation includes a significant change in transcription or translation level of at least 1.5-fold, in some embodiments at least two-fold, and in some embodiments at least five fold.

In certain embodiments, conditions that increase one or more cellular DNA repair processes may include one or more of: introduction of one or more sites into the GRON or into the plant cell DNA that are targets for base excision repair, introduction of one or more sites into the GRON or into the plant cell DNA that are targets for non-homologous end joining, introduction of one or more sites into the GRON or into the plant cell DNA that are targets for microhomology-mediated end joining, introduction of one or more sites into the GRON or into the plant cell DNA that are targets for homologous recombination, and introduction of one or more sites into the GRON or into the plant cell DNA that are targets for effecting repair (e.g., base-excision repair (BER); homologous recombination repair (HR); mismatch repair (MMR); non-homologous end-joining repair (NHEJ) which include classical and alternative NHEJ; and nucleotide excision repair (NER)).

As described herein, GRONs for use herein may include one or more of the following (non-limiting) alterations from conventional RNA and DNA nucleotides:

-   -   one or more abasic nucleotides;     -   one or more 8′oxo dA and/or 8′oxo dG nucleotides;     -   a reverse base at the 3′ end thereof,     -   one or more 2′O-methyl nucleotides;     -   one or more RNA nucleotides;     -   one or more RNA nucleotides at the 5′ end thereof, and in some         embodiments 2, 3, 4, 5, 6, 7, 8, 9, 10, or more; wherein one or         more of the RNA nucleotides may further be modified; one or more         RNA nucleotides at the 3′ end thereof, and in some embodiments         2, 3, 4, 5, 6, 7, 8, 9, 10, or more; wherein one or more of the         RNA nucleotides may further be modified;     -   one or more 2′O-methyl RNA nucleotides at the 5′ end thereof,         and in some embodiments 2, 3, 4, 5, 6, 7, 8, 9, 10, or more;     -   an intercalating dye;     -   a 5′ terminus cap;     -   a backbone modification selected from the group consisting of a         phosphothioate modification, a methyl phosphonate modification,         a locked nucleic acid (LNA) modification, a O-(2-methoxyethyl)         (MOE) modification, a di PS modification, and a peptide nucleic         acid (PNA) modification;     -   one or more intrastrand crosslinks;     -   one or more fluorescent dyes conjugated thereto, and in some         embodiments at the 5′ or 3′ end of the GRON; and     -   one or more bases which increase hybridization energy.

The term “wobble base” as used herein refers to a change in a one or more nucleotide bases of a reference nucleotide sequence wherein the change does not change the sequence of the amino acid coded by the nucleotide relative to the reference sequence.

The term “non-nucleotide” or “abasic nucleotide” as use herein refers to any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine. It may have substitutions for a 2′ or 3′ H or OH as described in the art and herein.

As described herein, in certain embodiments GRON quality and conversion efficiency may be improved by synthesizing all or a portion of the GRON using nucleotide multimers, such as dimers, trimers, tetramers, etc. improving its purity.

In certain embodiments, the target deoxyribonucleic acid (DNA) sequence is within a plant cell, for example the target DNA sequence is in the plant cell genome. The plant cell may be non-transgenic or transgenic, and the target DNA sequence may be a transgene or an endogenous gene of the plant cell.

In certain embodiments, the conditions that increase one or more cellular DNA repair processes comprise introducing one or more compounds which induce single or double DNA strand breaks into the plant cell prior to, or coincident to, or after delivering the GRON into the plant cell. Exemplary compounds are described herein.

In certain embodiments, the methods further comprise regenerating a plant having a mutation introduced by the GRON from the plant cell, and may comprise collecting seeds from the plant.

In related aspects, the present disclosure relates to plant cells comprising a genomic modification introduced by a GRON according to the methods described herein, a plant comprising a genomic modification introduced by a GRON according to the methods described herein, or a seed comprising a genomic modification introduced by a GRON according to the methods described herein; or progeny of a seed comprising a genomic modification introduced by a GRON according to the methods described herein.

Constructs

The nucleic acid molecules disclosed herein (e.g., site specific nucleases, or guide RNA for CRISPRs) can be used in the production of recombinant nucleic acid constructs. In one embodiment, the nucleic acid molecules of the present disclosure can be used in the preparation of nucleic acid constructs, for example, expression cassettes for expression in the plant, microorganism, or animal of interest such as SHP expression constructs optionally having one or more mutations as described herein. This expression may be transient for instance when the construct is not integrated into the host genome or maintained under the control offered by the promoter and the position of the construct within the host's genome if it becomes integrated.

Expression cassettes may include regulatory sequences operably linked to the site-specific nuclease or guide RNA sequences disclosed herein. The cassette may additionally contain at least one additional gene to be co-transformed into the organism. Alternatively, the additional gene or genes can be provided on multiple expression cassettes.

The nucleic acid constructs may be provided with a plurality of restriction sites for insertion of the site-specific nuclease coding sequence to be under the transcriptional regulation of the regulatory regions. The nucleic acid constructs may additionally contain nucleic acid molecules encoding for selectable marker genes.

Any promoter can be used in the production of the nucleic acid constructs. The promoter may be native or analogous, or foreign or heterologous, to the plant, microbial, or animal host nucleic acid sequences disclosed herein. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. Where the promoter is “foreign” or “heterologous” to the plant, microbial, or animal host, it is intended that the promoter is not found in the native plant, microbial, or animal into which the promoter is introduced. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.

The site directed nuclease sequences disclosed herein may be expressed using heterologous promoters.

Any promoter can be used in the preparation of constructs to control the expression of the site directed nuclease sequences, such as promoters providing for constitutive, tissue-preferred, inducible, or other promoters for expression in plants, microbes, or animals. Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43 838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al., Nature 313:810-812; 1985); rice actin (McElroy et al., Plant Cell 2:163-171, 1990); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632, 1989 and Christensen et al., Plant Mol. Biol. 18:675-689, 1992); pEMU (Last et al., Theor. Appl. Genet. 81:581-588, 1991); MAS (Velten et al., EMBO J. 3:2723-2730, 1984); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Tissue-preferred promoters can be utilized to direct site directed nuclease expression within a particular plant tissue. Such tissue-preferred promoters include, but are not limited to, leaf-preferred promoters, root-preferred promoters, seed-preferred promoters, and stem-preferred promoters. Tissue-preferred promoters include Yamamoto et al., Plant J. 12(2):255-265, 1997; Kawamata et al., Plant Cell Physiol. 38(7):792-803, 1997; Hansen et al., Mol. Gen Genet. 254(3):337-343, 1997; Russell et al., Transgenic Res. 6(2):157-168, 1997; Rinehart et al., Plant Physiol. 1 12(3):1331-1341, 1996; Van Camp et al., Plant Physiol. 1 12(2):525-535, 1996; Canevascini et al., Plant Physiol. 112(2): 513-524, 1996; Yamamoto et al., Plant Cell Physiol. 35(5):773-778, 1994; Lam, Results Probl. Cell Differ. 20:181-196, 1994; Orozco et al. Plant Mol Biol. 23(6):1129-1138, 1993; Matsuoka et al., Proc Nat'l. Acad. Sci. USA 90(20):9586-9590, 1993; and Guevara-Garcia et al., Plant J. 4(3):495-505, 1993.

The nucleic acid constructs may also include transcription termination regions. Where transcription terminations regions are used, any termination region may be used in the preparation of the nucleic acid constructs. For example, the termination region may be derived from another source (i.e., foreign or heterologous to the promoter). Examples of termination regions that are available for use in the constructs of the present disclosure include those from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al., Mol. Gen. Genet. 262:141-144, 1991; Proudfoot, Cell 64:671-674, 1991; Sanfacon et al., Genes Dev. 5:141-149, 1991; Mogen et al., Plant Cell 2:1261-1272, 1990; Munroe et al., Gene 91:151-158, 1990; Ballas et al., Nucleic Acids Res. 17:7891-7903, 1989; and Joshi et al., Nucleic Acid Res. 15:9627-9639, 1987.

In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, the nucleic acids may be optimized for increased expression in the transformed plant. That is, the nucleic acids encoding the site directed nuclease proteins can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri, (Plant Physiol. 92:1-11, 1990) for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al., Nucleic Acids Res. 17:477-498, 1989. See also e.g., Lanza et al., BMC Systems Biology 8:33-43, 2014; Burgess-Brown et al., Protein Expr. Purif. 59:94-102, 2008; Gustafsson et al., Trends Biotechnol 22:346-353, 2004.

In addition, other sequence modifications can be made to the nucleic acid sequences disclosed herein. For example, additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may also be adjusted to levels average for a target cellular host, as calculated by reference to known genes expressed in the host cell. In addition, the sequence can be modified to avoid predicted hairpin secondary mRNA structures.

Other nucleic acid sequences may also be used in the preparation of the constructs of the present disclosure, for example to enhance the expression of the site directed nuclease coding sequence. Such nucleic acid sequences include the introns of the maize AdhI, intron1 gene (Callis et al., Genes and Development 1:1183-1200, 1987), and leader sequences, (W-sequence) from the Tobacco Mosaic virus (TMV), Maize Chlorotic Mottle Virus and Alfalfa Mosaic Virus (Gallie et al., Nucleic Acid Res. 15:8693-8711, 1987; and Skuzeski et al., Plant Mol. Biol. 15:65-79, 1990). The first intron from the shrunken-1 locus of maize has been shown to increase expression of genes in chimeric gene constructs. U.S. Pat. Nos. 5,424,412 and 5,593,874 disclose the use of specific introns in gene expression constructs, and Gallie et al. (Plant Physiol. 106:929-939, 1994) also have shown that introns are useful for regulating gene expression on a tissue specific basis. To further enhance or to optimize site directed nuclease gene expression, the plant expression vectors disclosed herein may also contain DNA sequences containing matrix attachment regions (MARs). Plant cells transformed with such modified expression systems, then, may exhibit overexpression or constitutive expression of a nucleotide sequence of the disclosure.

The expression constructs disclosed herein can also include nucleic acid sequences capable of directing the expression of the site directed nuclease sequence to the chloroplast or other organelles and structures in both prokaryotes and eukaryotes. Such nucleic acid sequences include chloroplast targeting sequences that encodes a chloroplast transit peptide to direct the gene product of interest to plant cell chloroplasts. Such transit peptides are known in the art. With respect to chloroplast-targeting sequences, “operably linked” means that the nucleic acid sequence encoding a transit peptide (i.e., the chloroplast-targeting sequence) is linked to the site directed nuclease nucleic acid molecules disclosed herein such that the two sequences are contiguous and in the same reading frame. See, for example, Von Heijne et al., Plant Mol. Biol. Rep. 9:104-126, 1991; Clark et al., J. Biol. Chem. 264:17544-17550, 1989; Della-Cioppa et al., Plant Physiol. 84:965-968, 1987; Romer et al., Biochem. Biophys. Res. Commun. 196:1414-1421, 1993; and Shah et al., Science 233:478-481, 1986.

Chloroplast targeting sequences are known in the art and include the chloroplast small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al., Plant Mol. Biol. 30:769-780, 1996; Schnell et al., J. Biol. Chem. 266(5):3335-3342, 1991); 5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al., J. Bioenerg. Biomemb. 22(6):789-810, 1990); tryptophan synthase (Zhao et al., J. Biol. Chem. 270(1 1):6081-6087, 1995); plastocyanin (Lawrence et al., J. Biol. Chem. 272(33):20357-20363, 1997); chorismate synthase (Schmidt et al., J. Biol. Chem. 268(36):27447-27457, 1993); and the light harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al., J. Biol. Chem. 263:14996-14999, 1988). See also Von Heijne et al., Plant Mol. Biol. Rep. 9:104-126, 1991; Clark et al., J. Biol. Chem. 264:17544-17550, 1989; Della-Cioppa et al., Plant Physiol. 84:965-968, 1987; Romer et al., Biochem. Biophys. Res. Commun. 196:1414-1421, 1993; and Shah et al., Science 233: 478-481, 1986.

In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, the nucleic acid constructs may be prepared to direct the expression of the mutant site directed nuclease coding sequence from the plant cell chloroplast. Methods for transformation of chloroplasts are known in the art. See, for example, Svab et al., Proc. Nat'l. Acad. Sci. USA 87:8526-8530, 1990; Svab and Maliga, Proc. Nat'l. Acad. Sci. USA 90:913-917, 1993; Svab and Maliga, EMBO J. 12:601-606, 1993. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. Proc. Nat'l. Acad. Sci. USA 91:7301-7305, 1994.

The nucleic acids of interest to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the nucleic acids of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831, herein incorporated by reference.

The nucleic acid constructs can be used to transform plant cells and regenerate transgenic plants comprising the site directed nuclease coding sequences. Numerous plant transformation vectors and methods for transforming plants are available. See, for example, U.S. Pat. No. 6,753,458, An, G. et al., Plant Physiol., 81:301-305, 1986; Fry, J. et al., Plant Cell Rep. 6:321-325, 1987; Block, M., Theor. Appl Genet. 76:767-774, 1988; Hinchee et al., Stadler. Genet. Symp. 203212.203-212, 1990; Cousins et al., Aust. J. Plant Physiol. 18:481-494, 1991; Chee, P. P. and Slightom, J. L., Gene. 118:255-260, 1992; Christou et al., Trends. Biotechnol. 10:239-246, 1992; D'Halluin et al., Bio/Technol. 10:309-3 14, 1992; Dhir et al., Plant Physiol. 99:81-88, 1992; Casas et al., Proc. Nat'l. Acad Sci. USA 90:11212-11216, 1993; Christou, P., In Vitro Cell. Dev. Biol.-Plant 29P:1 19-124, 1993; Davies et al., Plant Cell Rep. 12:180-183, 1993; Dong, J. A. and Mc Hughen, A., Plant Sci. 91:139-148, 1993; Franklin, C. I., Trieu, T. N., Cassidy, B. G., Dixon, R. A., Nelson, R. S. 1993, Plant Cell Report 12, 74-79; Golovkin et al., Plant Sci. 90:41-52, 1993; Guo Chin Sci. Bull. 38:2072-2078; Asano et al., Plant Cell Rep. 13, 1994; Ayeres, N. M. and Park, W. D., Crit. Rev. Plant. Sci. 13:219-239, 1994; Barcelo et al., Plant. J. 5:583-592, 1994; Becker et al., Plant. J. 5:299-307, 1994; Borkowska et al., Acta. Physiol Plant. 16:225-230, 1994; Christou, P., Agro. Food. Ind. Hi Tech. 5:17-27, 1994; Eapen et al., Plant Cell Rep. 13:582-586, 1994; Hartman et al., Bio-Technology 12:919923, 1994; Ritala et al., Plant. Mol. Biol. 24:317-325, 1994; and Wan, Y. C. and Lemaux, P. G., Plant Physiol. 104:3748, 1994. The constructs may also be transformed into plant cells using homologous recombination.

The term “wild-type” when made in reference to a peptide sequence and nucleotide sequence refers to a peptide sequence and nucleotide sequence (locus/gene/allele), respectively, which has the characteristics of that peptide sequence and nucleotide sequence when isolated from a naturally occurring source. A wild-type peptide sequence and nucleotide sequence is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the peptide sequence and nucleotide sequence, respectively. “Wild-type” may also refer to the sequence at a specific nucleotide position or positions, or the sequence at a particular codon position or positions, or the sequence at a particular amino acid position or positions.

“Consensus sequence” is defined as a sequence of amino acids or nucleotides that contain identical amino acids or nucleotides or functionally equivalent amino acids or nucleotides for at least 25% of the sequence. The identical or functionally equivalent amino acids or nucleotides need not be contiguous.

A nucleobase is a base, which in certain preferred embodiments is a purine, pyrimidine, or a derivative or analog thereof. Nucleosides are nucleobases that contain a pentosefuranosyl moiety, e.g., an optionally substituted riboside or 2′-deoxyriboside. Nucleosides can be linked by one of several linkage moieties, which may or may not contain phosphorus. Nucleosides that arc linked by unsubstituted phosphodiester linkages are termed nucleotides. The term “nucleobase” as used herein includes peptide nucleobases, the subunits of peptide nucleic acids, and morpholine nucleobases as well as nucleosides and nucleotides.

An oligonucleobase is a polymer comprising nucleobases; in some embodiments at least, a portion of which can hybridize by Watson-Crick base pairing to a DNA having the complementary sequence. An oligonucleobase chain may have a single 5′ and 3′ terminus, which are the ultimate nucleobases of the polymer. A particular oligonucleobase chain can contain nucleobases of all types. An oligonucleobase compound is a compound comprising one or more oligonucleobase chains that may be complementary and hybridized by Watson-Crick base pairing. Ribo-type nucleobases include pentosefuranosyl containing nucleobases wherein the 2′ carbon is a methylene substituted with a hydroxyl, alkyloxy or halogen. Deoxyribo-type nucleobases are nucleobases other than ribo-type nucleobases and include all nucleobases that do not contain a pentosefuranosyl moiety.

In certain embodiments, an oligonucleobase strand may include both oligonucleobase chains and segments or regions of oligonucleobase chains. An oligonucleobase strand may have a 3′ end and a 5′ end, and when an oligonucleobase strand is coextensive with a chain, the 3′ and 5′ ends of the strand are also 3′ and 5′ termini of the chain.

As used herein the term “codon” refers to a sequence of three adjacent nucleotides (either RNA or DNA) constituting the genetic code that determines the insertion of a specific amino acid in a polypeptide chain during protein synthesis or the signal to stop protein synthesis. The term “codon” is also used to refer to the corresponding (and complementary) sequences of three nucleotides in the messenger RNA into which the original DNA is transcribed.

As used herein, the term “homology” refers to sequence similarity among proteins and DNA. The term “homology” or “homologous” refers to a degree of identity. There may be partial homology or complete homology. A partially homologous sequence is one that has less than 100% sequence identity when compared to another sequence.

“Heterozygous” refers to having different alleles at one or more genetic loci in homologous chromosome segments. As used herein “heterozygous” may also refer to a sample, a cell, a cell population or an organism in which different alleles at one or more genetic loci may be detected. Heterozygous samples may also be determined via methods known in the art such as, for example, nucleic acid sequencing. For example, if a sequencing electropherogram shows two peaks at a single locus and both peaks are roughly the same size, the sample may be characterized as heterozygous. Or, if one peak is smaller than another, but is at least about 25% the size of the larger peak, the sample may be characterized as heterozygous. In some embodiments, the smaller peak is at least about 15% of the larger peak. In other embodiments, the smaller peak is at least about 10% of the larger peak. In other embodiments, the smaller peak is at least about 5% of the larger peak. In other embodiments, a minimal amount of the smaller peak is detected.

As used herein, “homozygous” refers to having identical alleles at one or more genetic loci in homologous chromosome segments. “Homozygous” may also refer to a sample, a cell, a cell population or an organism in which the same alleles at one or more genetic loci may be detected. Homozygous samples may be determined via methods known in the art, such as, for example, nucleic acid sequencing. For example, if a sequencing electropherogram shows a single peak at a particular locus, the sample may be termed “homozygous” with respect to that locus.

The term “hemizygous” refers to a gene or gene segment being present only once in the genotype of a cell or an organism because the second allele is deleted, or is not present on the homologous chromosome segment. As used herein “hemizygous” may also refer to a sample, a cell, a cell population or an organism in which an allele at one or more genetic loci may be detected only once in the genotype.

The term “zygosity status” as used herein refers to a sample, a cell population, or an organism as appearing heterozygous, homozygous, or hemizygous as determined by testing methods known in the art and described herein. The term “zygosity status of a nucleic acid” means determining whether the source of nucleic acid appears heterozygous, homozygous, or hemizygous. The “zygosity status” may refer to differences in at a single nucleotide position in a sequence. In some methods, the zygosity status of a sample with respect to a single mutation may be categorized as homozygous wild-type, heterozygous (i.e., one wild-type allele and one mutant allele), homozygous mutant, or hemizygous (i.e., a single copy of either the wild-type or mutant allele).

As used herein, the term “RTDS” refers to The Rapid Trait Development System™ (RTDS™) developed by Cibus. RTDS is a site-specific gene modification system that is effective at making precise changes in a gene sequence without the incorporation of foreign genes or control sequences.

The term “about” as used herein means in quantitative terms plus or minus 10%. For example, “about 3%” would encompass 2.7-3.3% and “about 10%” would encompass 9-11%. Moreover, where “about” is used herein in conjunction with a quantitative term it is understood that in addition to the value plus or minus 10%, the exact value of the quantitative term is also contemplated and described. For example, the term “about 3%” expressly contemplates, describes and includes exactly 3%.

RTDS and Repair Oligonucleotides (GRONs)

Various aspects and embodiments of the methods and compositions contemplated herein include methods to improve the efficiency of the targeting of modifications to specific locations in genomic or other nucleotide sequences (for example modifications to an SHP gene such as contemplated herein).

RTDS in some embodiments is based on altering a targeted gene by utilizing the cell's own gene repair system to specifically modify the gene sequence in situ and not insert foreign DNA and gene expression control sequences. This procedure can effect a precise change in the genetic sequence while the rest of the genome is left unaltered. In some embodiments, in contrast to conventional transgenic GMOs, there is no integration of foreign genetic material, nor is any foreign genetic material left in the plant. The changes in the genetic sequence introduced by RTDS are not randomly inserted. Since affected genes remain in their native location, no random, uncontrolled or adverse pattern of expression occurs.

The molecule that effects this change is a chemically synthesized oligonucleotide (GRON) as described herein which may be composed of both DNA and modified RNA bases as well as other chemical moieties, and is designed to hybridize at the targeted gene location to create a mismatched base-pair. This mismatched base-pair acts as a signal to attract the cell's own natural gene repair system to that site and correct (replace, insert or delete) the designated nucleotide or nucleotides within the gene. Once the correction process is complete the GRON molecule is degraded and the now-modified or repaired gene is expressed under that gene's normal endogenous control mechanisms.

The methods and compositions disclosed herein can be practiced or made with “gene repair oligonucleobases” (GRON) having the conformations and chemistries as described in detail herein and below. The “gene repair oligonucleobases” as contemplated herein have also been described in published scientific and patent literature using other names including “recombinagenic oligonucleobases;” “RNA/DNA chimeric oligonucleotides;” “chimeric oligonucleotides;” “mixed duplex oligonucleotides” (MDONs); “RNA DNA oligonucleotides (RDOs);” “gene targeting oligonucleotides;” “genoplasts;” “single stranded modified oligonucleotides;” “Single stranded oligodeoxynucleotide mutational vectors” (SSOMVs); “duplex mutational vectors;” and “heteroduplex mutational vectors.” The gene repair oligonucleobase can be introduced into a plant cell using any method commonly used in the art, including but not limited to, microcarriers (biolistic delivery), microfibers, polyethylene glycol (PEG)-mediated uptake, electroporation, and microinjection.

In one embodiment, the gene repair oligonucleobase is a mixed duplex oligonucleotides (MDON) in which the RNA-type nucleotides of the mixed duplex oligonucleotide are made RNase resistant by replacing the 2′-hydroxyl with a fluoro, chloro or bromo functionality or by placing a substituent on the 2′-O. Suitable substituents include the substituents taught by the Kmiec II. Alternative substituents include the substituents taught by U.S. Pat. No. 5,334,711 (Sproat) and the substituents taught by patent publications EP 629 387 and EP 679 657 (collectively, the Martin Applications), which are hereby incorporated by reference. As used herein, a 2′-fluoro, chloro or bromo derivative of a ribonucleotide or a ribonucleotide having a T-OH substituted with a substituent described in the Martin Applications or Sproat is termed a “T-substituted ribonucleotide.” As used herein the term “RNA-type nucleotide” means a T-hydroxyl or 2′-substituted nucleotide that is linked to other nucleotides of a mixed duplex oligonucleotide by an unsubstituted phosphodiester linkage or any of the non-natural linkages taught by Kmiec I or Kmiec II. As used herein the term “deoxyribo-type nucleotide” means a nucleotide having a T-H, which can be linked to other nucleotides of a gene repair oligonucleobase by an unsubstituted phosphodiester linkage or any of the non-natural linkages taught by Kmiec I or Kmiec II.

In particular embodiments of the present disclosure, the gene repair oligonucleobase may be a mixed duplex oligonucleotide (MDON) that is linked solely by unsubstituted phosphodiester bonds. In alternative embodiments, the linkage is by substituted phosphodiesters, phosphodiester derivatives and non-phosphorus-based linkages as taught by Kmiec II. In yet another embodiment, each RNA-type nucleotide in the mixed duplex oligonucleotide is a 2′-Substituted Nucleotide. Particular preferred embodiments of 2′-Substituted Ribonucleotides are 2′-fluoro, T-methoxy, 2′-propyloxy, 2′-allyloxy, 2′-hydroxylethyloxy, 2′-methoxyethyloxy, T-fluoropropyloxy and 2′-trifluoropropyloxy substituted ribonucleotides. More preferred embodiments of 2′-Substituted Ribonucleotides are 2′-fluoro, 2′-methoxy, 2′-methoxyethyloxy, and 2′-allyloxy substituted nucleotides. In another embodiment the mixed duplex oligonucleotide is linked by unsubstituted phosphodiester bonds.

Although mixed duplex oligonucleotides (MDONs) having only a single type of 2′-substituted RNA-type nucleotide are more conveniently synthesized, the methods of the disclosure can be practiced with mixed duplex oligonucleotides having two or more types of RNA-type nucleotides. The function of an RNA segment may not be affected by an interruption caused by the introduction of a deoxynucleotide between two RNA-type trinucleotides, accordingly, the term RNA segment encompasses terms such as “interrupted RNA segment.” An uninterrupted RNA segment is termed a contiguous RNA segment. In an alternative embodiment an RNA segment can contain alternating RNase-resistant and unsubstituted 2′-OH nucleotides. The mixed duplex oligonucleotides in some embodiments have fewer than 100 nucleotides and other embodiments fewer than 85 nucleotides, but more than 50 nucleotides. The first and second strands are Watson-Crick base paired. In one embodiment the strands of the mixed duplex oligonucleotide are covalently bonded by a linker, such as a single stranded hexa, penta or tetranucleotide so that the first and second strands are segments of a single oligonucleotide chain having a single 3′ and a single 5′ end. The 3′ and 5′ ends can be protected by the addition of a “hairpin cap” whereby the 3′ and 5′ terminal nucleotides are Watson-Crick paired to adjacent nucleotides. A second hairpin cap can, additionally, be placed at the junction between the first and second strands distant from the 3′ and 5′ ends, so that the Watson-Crick pairing between the first and second strands is stabilized.

The first and second strands contain two regions that are homologous with two fragments of the target gene/allele, i.e., have the same sequence as the target gene/allele. A homologous region contains the nucleotides of an RNA segment and may contain one or more DNA-type nucleotides of connecting DNA segment and may also contain DNA-type nucleotides that are not within the intervening DNA segment. The two regions of homology are separated by, and each is adjacent to, a region having a sequence that differs from the sequence of the target gene, termed a “heterologous region.” The heterologous region can contain one, two or three mismatched nucleotides. The mismatched nucleotides can be contiguous or alternatively can be separated by one or two nucleotides that are homologous with the target gene/allele. Alternatively, the heterologous region can also contain an insertion or one, two, three or of five or fewer nucleotides. Alternatively, the sequence of the mixed duplex oligonucleotide may differ from the sequence of the target gene/allele only by the deletion of one, two, three, or five or fewer nucleotides from the mixed duplex oligonucleotide. The length and position of the heterologous region is, in this case, deemed to be the length of the deletion, even though no nucleotides of the mixed duplex oligonucleotide are within the heterologous region. The distance between the fragments of the target gene that are complementary to the two homologous regions is identical to the length of the heterologous region where a substitution or substitutions is intended. When the heterologous region contains an insertion, the homologous regions are thereby separated in the mixed duplex oligonucleotide farther than their complementary homologous fragments are in the gene/allele, and the converse is applicable when the heterologous region encodes a deletion.

The RNA segments of the mixed duplex oligonucleotides are each a part of a homologous region, i.e., a region that is identical in sequence to a fragment of the target gene, which segments together in some embodiments contain at least 13 RNA-type nucleotides and in some embodiments from 16 to 25 RNA-type nucleotides or yet other embodiments 18-22 RNA-type nucleotides or in some embodiments 20 nucleotides. In one embodiment, RNA segments of the homology regions are separated by and adjacent to, i.e., “connected by” an intervening DNA segment. In one embodiment, each nucleotide of the heterologous region is a nucleotide of the intervening DNA segment. An intervening DNA segment that contains the heterologous region of a mixed duplex oligonucleotide is termed a “mutator segment.”

In another embodiment of the methods and compositions of the present disclosure, a gene repair oligonucleobase (GRON) is a single stranded oligodeoxynucleotide mutational vector (SSOMV), such as disclosed in International Patent Application PCT/USOO/23457, U.S. Pat. Nos. 6,271,360, 6,479,292, and 7,060,500 which is incorporated by reference in its entirety. The sequence of the SSOMV is based on the same principles as the mutational vectors described in U.S. Pat. Nos. 5,756,325; 5,871,984; 5,760,012; 5,888,983; 5,795,972; 5,780,296; 5,945,339; 6,004,804; and 6,010,907 and in International Publication Nos. WO 98/49350; WO 99/07865; WO 99/58723; WO 99/58702; and WO 99/40789. The sequence of the SSOMV contains two regions that are homologous with the target sequence separated by a region that contains the desired genetic alteration termed the mutator region. The mutator region can have a sequence that is the same length as the sequence that separates the homologous regions in the target sequence, but having a different sequence. Such a mutator region can cause a substitution. Alternatively, the homologous regions in the SSOMV can be contiguous to each other, while the regions in the target gene having the same sequence are separated by one, two or more nucleotides. Such an SSOMV causes a deletion from the target gene of the nucleotides that are absent from the SSOMV. Lastly, the sequence of the target gene that is identical to the homologous regions may be adjacent in the target gene but separated by one, two, or more nucleotides in the sequence of the SSOMV. Such an SSOMV causes an insertion in the sequence of the target gene. In certain embodiments, a SSOMV does not anneal to itself.

The nucleotides of the SSOMV are deoxyribonucleotides that are linked by unmodified phosphodiester bonds except that the 3′ terminal and/or 5′ terminal internucleotide linkage or alternatively the two 3′ terminal and/or 5′ terminal internucleotide linkages can be a phosphorothioate or phosphoamidate. As used herein an internucleotide linkage is the linkage between nucleotides of the SSOMV and does not include the linkage between the 3′ end nucleotide or 5′ end nucleotide and a blocking substituent. In a specific embodiment the length of the SSOMV is between 21 and 55 deoxynucleotides and the lengths of the homology regions are, accordingly, a total length of at least 20 deoxynucleotides and at least two homology regions should each have lengths of at least 8 deoxynucleotides.

The SSOMV can be designed to be complementary to either the coding or the non-coding strand of the target gene. When the desired mutation is a substitution of a single base, it is preferred that both the mutator nucleotide and the targeted nucleotide be a pyrimidine. To the extent that is consistent with achieving the desired functional result, it is preferred that both the mutator nucleotide and the targeted nucleotide in the complementary strand be pyrimidines. Particularly preferred are SSOMVs that encode transversion mutations, i.e., a C or T mutator nucleotide is mismatched, respectively, with a C or T nucleotide in the complementary strand.

2′-OME GRON Design. In various embodiments, a GRON may have both RNA and DNA nucleotides and/or other types of nucleobases. In some embodiments, one or more of the DNA or RNA nucleotides comprise a modification. In certain embodiments, the first 5′ nucleotide is an RNA nucleotide and the remainder of the nucleotides are DNA. In still further embodiments, the first 5′ RNA nucleotide is modified with a 2-O-Me. In other embodiments, the first two, three, four, five, six, seven, eight, nine, ten or more 5′ nucleotides are an RNA nucleotide and the remainder of the nucleotides are DNA. In still further embodiments, one or more of the first two, three, four, five, six, seven, eight, nine, ten or more 5′ RNA nucleotide are modified with a 2′-O-Me. In plant cells, double-strand beaks in DNA are typically repaired by the NHEJ DNA repair pathway. This pathway does not require a template to repair the DNA and is therefore error prone. The advantage of using this pathway to repair DNA for a plant cell is that it is quick, ubiquitous and most importantly can occur at times when a cell is not undergoing DNA replication. Another DNA repair pathway that functions in repairing double-strand breaks outside of the replication fork in plant cells is called templated repair; however, unlike the NHEJ pathway this type of repair is precise and requires the use of a DNA template (GRON).

Improving Efficiency

The present disclosure may include any of a number of approaches to increase the effectiveness of conversion of a target gene using repair oligonucleotides, and which may be used alone or in combination with one another. These include, for example:

-   -   1. Introducing modifications to the repair oligonucleotides         which attract DNA repair machinery to the targeted (mismatch)         site.         -   A. Introduction of one or more abasic sites in the             oligonucleotide (e.g., within 10 bases, and in some             embodiments with 5 bases of the desired mismatch site)             generates a lesion which is an intermediate in base excision             repair (BER), and which attracts BER machinery to the             vicinity of the site targeted for conversion by the repair             oligonucleotide. dSpacer (abasic furan) modified             oligonucleotides may be prepared as described in, for             example, Takeshita et al., J. Biol. Chem., 262:10171-79,             1987.         -   B. Inclusion of compounds which induce single or double             strand breaks, either into the oligonucleotide or together             with the oligonucleotide, generates a lesion which is             repaired by NHEJ, microhomology-mediated end joining (MMEJ),             and homologous recombination. By way of example, the             bleomycin family of antibiotics, zinc fingers, FokI (or any             type IIS class of restriction enzyme) and other nucleases             may be covalently coupled to the 3′ or 5′ end of repair             oligonucleotides, in order to introduce double strand breaks             in the vicinity of the site targeted for conversion by the             repair oligonucleotide. The bleomycin family of antibiotics             are DNA cleaving glycopeptides which include bleomycin,             zeocin, phleomycin, tallysomycin, pepleomycin and others.         -   C. Introduction of one or more 8′oxo dA or dG incorporated             in the oligonucleotide (e.g., within 10 bases, and in some             embodiments with 5 bases of the desired mismatch site)             generates a lesion which is similar to lesions created by             reactive oxygen species. These lesions induce the so-called             “pushing repair” system. See, e.g., Kim et al., J. Biochem.             Mol. Biol. 37:657-62, 2004.     -   2. Increase stability of the repair oligonucleotides:         -   Introduction of a reverse base (idC) at the 3′ end of the             oligonucleotide to create a 3′ blocked end on the repair             oligonucleotide.         -   Introduction of one or more 2′O-methyl nucleotides or bases             which increase hybridization energy (see, e.g.,             WO2007/073149) at the 5′ and/or 3′ of the repair             oligonucleotide.         -   Introduction of one or a plurality of 2′O-methyl RNA             nucleotides at the 5′ end of the repair oligonucleotide,             leading into DNA bases which provide the desired mismatch             site, thereby creating an Okazaki Fragment-like nucleic acid             structure.         -   Conjugated (5′ or 3′) intercalating dyes such as acridine,             psoralen, ethidium bromide and Syber stains.         -   Introduction of a 5′ terminus cap such as a T/A clamp, a             cholesterol moiety, SIMA (HEX), riboC and amidite.         -   Backbone modifications such as phosphothioate, 2′-0 methyl,             methyl phosphonates, locked nucleic acid (LNA), MOE             (methoxyethyl), di PS and peptide nucleic acid (PNA).         -   Crosslinking of the repair oligonucleotide, e.g., with             intrastrand crosslinking reagents agents such as cisplatin             and mitomycin C.         -   Conjugation with fluorescent dyes such as Cy3, DY547, Cy3.5,             Cy3B, Cy5 and DY647.     -   3. Increase hybridization energy of the repair oligonucleotide         through incorporation of bases which increase hybridization         energy (see, e.g., WO2007/073149).     -   4. Increase the quality of repair oligonucleotide synthesis by         using nucleotide multimers (dimers, trimers, tetramers, etc.) as         building blocks for synthesis. This results in fewer coupling         steps and easier separation of the full-length products from         building blocks.     -   5. Use of long repair oligonucleotides (i.e., greater than 55         nucleotides in length, for example such as the lengths described         herein, for example having one or more mutations or two or more         mutations targeted in the repair oligonucleotide.

Examples of the foregoing approaches are provided in Table A.

TABLE A Exemplary GRON chemistries Oligo type Modifications 5′ mods T/A clamp T/A clamp Backbone Phosphothioate PS modifications Intercalating dyes 5′ Acridine 3′ idC Acridine, idC 2′-O-methyl DNA/RNA Cy3 replacements DY547 Facilitators 2′-O-Me oligos designed 2′-O-Me 5′ and 3′ of the converting oligo Abasic Abasic site placed in Abasic 2 various locations 5′ and 3′ to the converting base. 44 mer Assist Assist approach Cy3, idC on one, none Overlap: on the other: 2 oligos: 1 with Cy3/idC, 1 unmodified repair oligo Assist Assist approach only make the No overlap: unmodified oligo 2 oligos: 1 with Cy3/idC, 1 unmodified repair oligo Abasic THF site placed in various Tetrahydrofuran locations 5′ and 3′ to the ( dspacer) converting base. 44 mer Backbone 9 2′-O-Me modifications Trimers Trimer amidites, Cy3. idC Pushing repair 8′oxo dA, 5′ Cy3, idC Pushing repair 8′oxo dA, 5′ Cy3, idC Double Strand Bleomycin Break Crosslinker Cisplatin Crosslinker Mitomycin C Facilitators super bases 5′ and 3′ of 2 amino dA and converting oligo 2-thio T Super oligos 2′amino d, 5′ Cy3, idC Super oligos 2-thio T, 5′ Cy3, idC Super oligos 7-deaza A, 5′ Cy3, idC Super oligos 7-deaza G, 5′ Cy3, idC Super oligos propanyl dC, 5′ Cy3, idC Intercalating dyes 5′ Psoralen/3′ idC Psoralen, idC Intercalating dyes 5′ Ethidium bromide Ethidium bromide Intercalating dyes 5′ Syber stains Syber stains 5′ mods 5′ Chol/3′ idC Cholesterol Double mutation Long oligo (55+ bases) w/ Any modification 2 mutation 5′ mods 5′ SIMA HEX/3′idC SIMA HEX, idC Backbone 9 Methyl phosphonates modifications Backbone LNA modifications Backbone 1 MOE (methoxyethyl) modifications Cy3 replacements Cy3.5 Cy3 replacements Cy5 Backbone di PS modifications 5′ mods riboC for branch nm Backbone PNA modifications Cy3 replacements DY647 5′ mods 5′ branch symmetric branch amidite/idC

The foregoing modifications may also include known nucleotide modifications such as methylation, 5′ intercalating dyes, modifications to the 5′ and 3′ ends, backbone modifications, crosslinkers, cyclization and ‘caps’ and substitution of one or more of the naturally occurring nucleotides with an analog such as inosine. Modifications of nucleotides include the addition of acridine, amine, biotin, cascade blue, cholesterol, Cy3@, Cy5@, Cy5.5@ Daboyl, digoxigenin, dinitrophenyl, Edans, 6-FAM., fluorescein, 3′-glyceryl, HEX, IRD-700, IRD-800, JOE, phosphate psoralen, rhodamine, ROX, thiol (SH), spacers, TAMRA, TET, AMCA-S”, SE, BODIPY^(∘), Marina Blue@, Pacific Blue@, Oregon Green@, Rhodamine Green@, Rhodamine Red@, Rhodol Green@ and Texas Red@. Polynucleotide backbone modifications include methylphosphonate, 2′-OMe-methylphosphonate RNA, phosphorothiorate, RNA, 2′-OMeRNA. Base modifications include 2-amino-dA, 2-aminopurine, 3′-(ddA), 3′dA (cordycepin), 7-deaza-dA, 8-Br-dA, 8-oxo-dA, N6-Me-dA, abasic site (dSpacer), biotin dT, 2′-OMe-5Me-C, 2′-OMe-propynyl-C, 3′-(5-Me-dC), 3′-(ddC), 5-Br-dC, 5-1-duc, 5-Me-dC, 5-F-dC, carboxy-dT, convertible dA, convertible dC, convertible dG, convertible dT, convertible dU, 7-deaza-dG, 8-Br-dG, 8-oxo-dG, O6-Me-dG, S6-DNP-dG, 4-methyl-indole, 5-nitroindole, 2′-OMe-inosine, 2′-dl, o6-phenyl-dl, 4-methyl-indole, 2′-deoxynebularine, 5-nitroindole, 2-aminopurine, dP (purine analogue), dK (pyrimidine analogue), 3-nitropyrrole, 2-thio-dT, 4-thio-dT, biotin-dT, carboxy-dT, 04-Me-dT, 04-triazol dT, 2′-OMe-propynyl-U, 5-Br-dU, 2′-dU, 5-F-dU, 5-1-dU, 04-triazol dU. Said terms also encompass peptide nucleic acids (PNAs), a DNA analogue in which the backbone is a pseudopeptide consisting of N-(2-aminoethyl)-glycine units rather than a sugar. PNAs mimic the behavior of DNA and bind complementary nucleic acid strands. The neutral backbone of PNA results in stronger binding and greater specificity than normally achieved. In addition, the unique chemical, physical and biological properties of PNA have been exploited to produce powerful biomolecular tools, antisense and antigene agents, molecular probes and biosensors.

Oligonucleobases may have nick(s), gap(s), modified nucleotides such as modified oligonucleotide backbones, abasic nucleotides, or other chemical moieties. In a further embodiment, at least one strand of the oligonucleobase includes at least one additional modified nucleotide, e.g., a 2′-O-methyl modified nucleotide such as a MOE (methoxyethyl), a nucleotide having a 5′-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide (the nucleobase is missing or has a hydroxyl group in place thereof (see, e.g., Glen Research, worldwide web address: glenresearch.com/GlenReports/GR21-14.html)), a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidite, and a non-natural base comprising nucleotide. Various salts, mixed salts and free acid forms are also included.

Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphoro-dithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl-phosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). The most common use of a linkage inversion is to add a 3′-3′ linkage to the end of an antisense oligonucleotide with a phosphorothioate backbone. The 3′-3′ linkage further stabilizes the antisense oligonucleotide to exonuclease degradation by creating an oligonucleotide with two 5′-OH ends and no 3′-OH end. Linkage inversions can be introduced into specific locations during oligonucleotide synthesis through use of “reversed phosphoramidites”. These reagents have the phosphoramidite groups on the 5′-OH position and the dimethoxytrityl (DMT) protecting group on the 3-OH position. Normally, the DMT protecting group is on the 5′-OH and the phosphoramidite is on the 3′-OH.

Examples of modified bases include, but are not limited to, 2-aminopurine, 2′-amino-butyryl pyrene-uridine, 2′-aminouridine, 2′-deoxyuridine, 2′-fluoro-cytidine, 2′-fluoro-uridine, 2,6-diaminopurine, 4-thio-uridine, 5-bromo-uridine, 5-fluoro-cytidine, 5-fluorouridine, 5-indo-uridine, 5-methyl-cytidine, inosine, N3-methyl-uridine, 7-deaza-guanine, 8-aminohexyl-amino-adenine, 6-thio-guanine, 4-thio-thymine, 2-thio-thymine, 5-iodo-uridine, 5-iodo-cytidine, 8-bromo-guanine, 8-bromo-adenine, 7-deaza-adenine, 7-diaza-guanine, 8-oxo-guanine, 5,6-dihydro-uridine, and 5-hydroxymethyl-uridine. These synthetic units are commercially available; (for example, purchased from Glen Research Company) and can be incorporated into DNA by chemical synthesis.

Examples of modification of the sugar moiety are 3′-deoxylation, 2′-fluorination, and arabanosidation, however, it is not to be construed as being limited thereto. Incorporation of these into DNA is also possible by chemical synthesis.

Examples of the 5′ end modification are 5′-amination, 5′-biotinylation, 5′-fluoresceinylation, 5′-tetrafluoro-fluoreceinyaltion, 5′-thionation, and 5′-dabsylation, however it is not to be construed as being limited thereto.

Examples of the 3′ end modification are 3′-amination, 3′-biotinylation, 2,3-dideoxidation, 3′-thionation, 3′-dabsylation, 3′-carboxylation, and 3′-cholesterylation, however, it is not to be construed as being limited thereto.

In one preferred embodiment, the oligonucleobase can contain a 5′ blocking substituent that is attached to the 5′ terminal carbons through a linker. The chemistry of the linker is not critical other than its length, which should in some embodiments be at least 6 atoms long and that the linker should be flexible. A variety of non-toxic substituents such as biotin, cholesterol or other steroids or a non-intercalating cationic fluorescent dye can be used. Particularly preferred reagents to make oligonucleobases are the reagents sold as Cy3™ and Cy5™ by Glen Research, Sterling Va. (now GE Healthcare), which are blocked phosphoroamidites that upon incorporation into an oligonucleotide yield 3,3,3′,3′-tetramethyl N,N′-isopropyl substituted indomonocarbocyanine and indodicarbocyanine dyes, respectively. Cy3 is particularly preferred. When the indocarbocyanine is N-oxyalkyl substituted it can be conveniently linked to the 5′ terminal of the oligodeoxynucleotide as a phosphodiester with a 5′ terminal phosphate. When the commercially available Cy3 phosphoramidite is used as directed, the resulting 5′ modification consists of a blocking substituent and linker together which are a N-hydroxypropyl, N′-phosphatidylpropyl 3,3,3′,3′-tetramethyl indomonocarbocyanine. Other dyes contemplated include Rhodamine6G, Tetramethylrhodamine, Sulforhodamine 101, Merocyanine 540, Atto565, Atto550 26, Cy3.5, Dy547, Dy548, Dy549, Dy554, Dy555, Dy556, Dy560, mStrawberry and mCherry.

In a preferred embodiment the indocarbocyanine dye is tetra substituted at the 3 and 3′ positions of the indole rings. Without limitations as to theory these substitutions prevent the dye from being an intercalating dye. The identity of the substituents at these positions is not critical.

The oligo designs herein described might also be used as more efficient donor templates in combination with other DNA editing or recombination technologies including, but not limited to, gene targeting using site-specific homologous recombination by zinc finger nucleases, meganucleases, Transcription Activator-Like Effector Nucleases (TALENs) or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs).

The present disclosure in certain aspects and embodiments may include methods and compositions relating to methods for the efficient modification of genomic cellular DNA and/or recombination of DNA into the genomic DNA of cells. Although not limited to any particular use, some methods provided herein may in certain embodiments be useful in, for example, introducing a modification into the genome of a cell for the purpose of determining the effect of the modification on the cell. For example, a modification may be introduced into the nucleotide sequence which encodes an enzyme to determine whether the modification alters the enzymatic activity of the enzyme, and/or determine the location of the enzyme's catalytic region. Alternatively, the modification may be introduced into the coding sequence of a DNA-binding protein to determine whether the DNA binding activity of the protein is altered, and thus to delineate the particular DNA-binding region within the protein. Yet another alternative is to introduce a modification into a non-coding regulatory sequence (e.g., promoter, enhancer, regulatory RNA sequence (miRNA), etc.) in order to determine the effect of the modification on the level of expression of a second sequence which is operably linked to the non-coding regulatory sequence. This may be desirable to, for example, define the particular sequence which possesses regulatory activity.

DNA Cutters

One strategy for producing targeted gene disruption is through the generation of single strand or double strand DNA breaks using a DNA cutter such as a site-specific endonuclease. Endonucleases are most often used for targeted gene disruption in organisms that have traditionally been refractive to more conventional gene targeting methods, such as algae, plants, and large animal models, including humans. For example, there are currently human clinical trials underway involving zinc finger nucleases for the treatment and prevention of HIV infection. Additionally, endonuclease engineering is currently being used in attempts to disrupt genes that produce undesirable phenotypes in crops.

Certain aspects of the present disclosure related to introducing one or more mutations into a targeted nucleic acid using a DNA endonuclease. In some embodiments, the DNA endonuclease is an RNA-guided DNA endonuclease. Exemplary RNA-guided DNA endonucleases include Cas9, Cpf1, and the like. RNA-guided DNA endonucleases suitable for use in the methods and compositions described herein will be readily apparent to one of skill in the art. Additional DNA endonucleases for use in the methods and compositions of the present disclosure are described herein.

Zinc Fingers

One class of artificial endonucleases is the zinc finger endonucleases. Zinc finger endonucleases combine a non-specific cleavage domain, typically that of FokI endonuclease, with zinc finger protein domains that are engineered to bind to specific DNA sequences. The modular structure of the zinc finger endonucleases makes them a versatile platform for delivering site-specific double-strand breaks to the genome. As FokI endonuclease cleaves as a dimer, one strategy to prevent off-target cleavage events has been to design zinc finger domains that bind at adjacent 9 base pair sites. See also U.S. Pat. Nos. 7,285,416; 7,521,241; 7,361,635; 7,273,923; 7,262,054; 7,220,719; 7,070,934; 7,013,219; 6,979,539; 6,933,113; 6,824,978; each of which is hereby herein incorporated by reference in its entirety.

TALENs

TALENs are targetable nucleases are used to induce single- and double-strand breaks into specific DNA sites, which are then repaired by mechanisms that can be exploited to create sequence alterations at the cleavage site.

The fundamental building block that is used to engineer the DNA-binding region of TALENs is a highly conserved repeat domain derived from naturally occurring TALEs encoded by Xanthomonas spp. proteobacteria. DNA binding by a TALEN is mediated by arrays of highly conserved 33-35 amino acid repeats that are flanked by additional TALE-derived domains at the amino-terminal and carboxy-terminal ends of the repeats.

These TALE repeats specifically bind to a single base of DNA, the identity of which is determined by two hypervariable residues typically found at positions 12 and 13 of the repeat, with the number of repeats in an array corresponded to the length of the desired target nucleic acid, the identity of the repeat selected to match the target nucleic acid sequence. In some embodiments, the target nucleic acid is between 15 and 20 base pairs in order to maximize selectivity of the target site. Cleavage of the target nucleic acid typically occurs within 50 base pairs of TALEN binding. Computer programs for TALEN recognition site design have been described in the art. See, e.g., Cermak et al., Nucleic Acids Res. 2011 July; 39(12): e82.

Once designed to match the desired target sequence, TALENs can be expressed recombinantly and introduced into protoplasts as exogenous proteins, or expressed from a plasmid within the protoplast or administered as mRNA or as protein.

Meganucleases

The homing endonucleases, also known as meganucleases, are sequence specific endonucleases that generate double strand breaks in genomic DNA with a high degree of specificity due to their large (e.g., >14 bp) cleavage sites. While the specificity of the homing endonucleases for their target sites allows for precise targeting of the induced DNA breaks, homing endonuclease cleavage sites are rare and the probability of finding a naturally occurring cleavage site in a targeted gene is low.

Another class of artificial endonucleases is the engineered meganucleases. Engineered homing endonucleases are generated by modifying the specificity of existing homing endonucleases. In one approach, variations are introduced in the amino acid sequence of naturally occurring homing endonucleases and then the resultant engineered homing endonucleases are screened to select functional proteins which cleave a targeted binding site. In another approach, chimeric homing endonucleases are engineered by combining the recognition sites of two different homing endonucleases to create a new recognition site composed of a half-site of each homing endonuclease. See e.g., U.S. Pat. Nos. 8,338,157, and 8,445,251.

CRISPRs or CRISPR/Cas Systems

CRISPR-Cas system contains three basic design components: 1) Cas gene, transcript (e.g., mRNA) or protein; 2) guide RNA (gRNA); and 3) crRNAs (CRISPR RNA) are RNA segments processed from RNA transcripts encoding the CRISPR repeat arrays, which harbor a “protospacer” region that are complementary to a foreign DNA site (e.g., endogenous DNA target region) and a part of the CRISPR repeat. See e.g., PCT Application Nos WO/2014/093661 and WO/2013/176772.

Cas (CRISPR Associated) Gene, Transcript (e.g., mRNA) or Protein

Transient Cas expression from a plasmid vector, direct delivery of Cas protein and or direct delivery of Cas mRNA into plant cells. Cas genes are codon optimized for expression in higher plants, algae or yeast and are driven by either a constitutive, inducible, tissue-specific or species-specific promoter when applicable. Cas transcript termination and polyadenlyation signals are either NosT, RBCT, HSP18.2T or other gene specific or species-specific terminators. Cas gene cassettes may contain introns, either native or in combination with gene-specific promoters and or synthetic promoters. Cas protein may contain one or more nuclear localization signal sequences (NLS), mutations, deletions, alterations or truncations. In transient expression systems, Cas gene cassettes may be combined with other components of the CRISPR-Cas system such as gRNA cassettes on the same transient expression vector. Alternatively, Cas gene cassettes may be located and expressed from constructs independent of gRNA cassettes or from other components of the CRISPR-Cas system. CRISPR associated (Cas) gene—encode for proteins with a variety of predicted nucleic acid-manipulating activities such as nucleases, helicases and polymerase. Cas genes include Cas9. Cas9 is a gene encoding a large protein containing a predicted RuvC-like and HNH endonuclease domains and is associated with the CRISPR adaptive immunity system that is present in most archaea and many bacteria. Cas9 protein consists of two lobes:

1) Recognition (REC) lobe—consists of three domains:

-   -   a) BH (bridge helix)     -   b) REC1—facilitates RNA-guided DNA targeting     -   c) REC2—facilitates RNA-guided DNA targeting

2) Nuclease (NUC) lobe—consists of three domains:

-   -   a) RuvC—facilitates RNA-guided DNA targeting; endonuclease         activity     -   b) HNH—endonuclease activity     -   c) PI—PAM interacting

In other embodiments, the Cas gene may be a homolog of Cas9 in which the RuvC, HNH, REC and BH domains are highly conserved. In some embodiments, Cas genes are those from the following species listed in Table B.

TABLE B Exemplary Cas Genes Locus ID/GI Species Cas profile ID Cas gene 352684361 Acidaminococcus_intestini_RyC_MR95_uid74445 mkCas0193 cas9 117929158 Acidothermus_cellulolyticus_11B_uid58501 cd09643 cas9 326315085 Acidovorax_avenae_ATCC_19860_uid42497 cd09643 cas9 222109285 Acidovorax_ebreus_TPSY_uid59233 COG3513 cas9 152978060 Actinobacillus_succinogenes_130Z_uid58247 COG3513 cas9 407692091 Actinobacillus_suis_H91_0380_uid176363 COG3513 cas9 187736489 Akkermansia_muciniphila_ATCC_BAA_835_uid58985 cd09643 cas9 319760940 Alicycliphilus_denitrificans_BC_uid49953 cd09643 cas9 330822845 Alicycliphilus_denitrificans_K601_uid66307 cd09643 cas9 288957741 Azospirillum_B510_uid46085 cd09643 cas9 549484339 Bacteroides_CF50_uid222805 cd09643, COG3513 cas9 375360193 Bacteroides_fragilis_638R_uid84217 COG3513, COG3513 cas9 60683389 Bacteroides_fragilis_NCTC_9343_uid57639 COG3513, COG3513 cas9 471261880 Bdellovibrio_exovorus_JSS_uid194119 COG3513 cas9 390944707 Belliella_baltica_DSM_15883_uid168182 cd09643, COG3513 cas9 470166767 Bibersteinia_trehalosi_192_uid193709 COG3513 cas9 310286728 Bifidobacterium_bifidum_S17_uid59545 mkCas0193 cas9 283456135 Bifidobacterium_dentium_Bd1_uid43091 cd09643 cas9 189440764 Bifidobacterium_longum_DJO10A_uid58833 cd09643 cas9 384200944 Bifidobacterium_longum_KACC_91563_uid158861 cd09643 cas9 479188345 Butyrivibrio_fibrisolvens_uid197155 cd09643 cas9 544063172 Campylobacter_jejuni_00_2425_uid219359 COG3513 cas9 543948719 Campylobacter_jejuni_00_2426_uid219324 COG3513 cas9 543946932 Campylobacter_jejuni_00_2538_uid219325 COG3513 cas9 543950499 Campylobacter_jejuni_00_2544_uid219326 COG3513 cas9 549693479 Campylobacter_jejuni_4031_uid222817 COG3513 cas9 157415744 Campylobacter_jejuni_81116_uid58771 COG3513 cas9 384448746 Campylobacter_jejuni_IA3902_uid159531 COG3513 cas9 384442102 Campylobacter_jejuni_M1_uid159535 COG3513 cas9 384442103 Campylobacter_jejuni_M1_uid159535 COG3513 cas9 403056243 Campylobacter_jejuni_NCTC_11168_BN148_uid174152 COG3513 cas9 218563121 Campylobacter_jejuni_NCTC_11168_ATCC_700819_uid57587 COG3513 cas9 407942868 Campylobacter_jejuni_PT14_uid176499 COG3513 cas9 153952471 Campylobacter_jejuni_doylei_269_97_uid58671 COG3513 cas9 294086111 Candidatus_Puniceispirillum_marinum_IMCC1322_uid47081 cd09643 cas9 340622236 Capnocytophaga_canimorsus_Cc5_uid70727 COG3513, cd09643 cas9 220930482 Clostridium_cellulolyticum_H10_uid58709 COG3513 cas9 479136975 Coprococcus_catus_GD_7_uid197174 mkCas0193 cas9 328956315 Coriobacterium_glomerans_PW2_uid65787 mkCas0193 cas9 375289763 Corynebacterium_diphtheriae_241_uid83607 cd09643 cas9 376283539 Corynebacterium_diphtheriae_31A_uid84309 cd09643 cas9 376286566 Corynebacterium_diphtheriae_BH8_uid84311 cd09643 cas9 376289243 Corynebacterium_diphtheriae_C7_beta_uid84313 cd09643 cas9 376244596 Corynebacterium_diphtheriae_HC01_uid84297 cd09643 cas9 376292154 Corynebacterium_diphtheriae_HC02_uid84317 cd09643 cas9 38232678 Corynebacterium_diphtheriae_NCTC_13129_uid57691 cd09643 cas9 376256051 Corynebacterium_diphtheriae_VA01_uid84305 cd09643 cas9 159042956 Dinoroseobacter_shibae_DFL_12_uid58707 cd09643 cas9 339445983 Eggerthella_YY7918_uid68707 mkCas0193 cas9 187250660 Elusimicrobium_minutum_Pei191_uid58949 cd09643 cas9 479180325 Enterococcus_7L76_uid197170 cd09643 cas9 397699066 Enterococcus_faecalis_D32_uid171261 mkCas0193 cas9 384512368 Enterococcus_faecalis_OG1RF_uid54927 mkCas0193 cas9 392988474 Enterococcus_hirae_ATCC_9790_uid70619 mkCas0193 cas9 558685081 Enterococcus_mundtii_QU_25_uid229420 mkCas0193 cas9 238924075 Eubacterium_rectale_ATCC_33656_uid59169 cd09643 cas9 385789535 Fibrobacter_succinogenes_S85_uid161919 cd09643, cd09643 cas9 261414553 Fibrobacter_succinogenes_S85_uid41169 cd09643, cd09643 cas9 374307738 Filifactor_alocis_ATCC_35896_uid46625 mkCas0193 cas9 169823755 Finegoldia_magna_ATCC_29328_uid58867 mkCas0193 cas9 150025575 Flavobacterium_psychrophilum_JIP02_86_uid61627 cd09643, cd09643 cas9 327405121 Fluviicola_taffensis_DSM_16823_uid65271 cd09643, cd09643 cas9 387824704 Francisella_cf_novicida_3523_uid162107 cd09704 cas9 118497352 Francisella_novicida_U112_uid58499 cd09704 cas9 134302318 Francisella_tularensis_WY96_3418_uid58811 cd09704 cas9 89256630 Francisella_tularensis_holarctica_LVS_uid58595 cd09704 cas9 89256631 Francisella_tularensis_holarctica_LVS_uid58595 cd09704 cas9 534508854 Fusobacterium_3_1_36A2_uid55995 mkCas0193 cas9 530600688 Geobacillus_JF8_uid215234 COG3513 cas9 209542524 Gluconacetobacter_diazotrophicus_PA1_5_uid59075 COG3513 cas9 162147907 Gluconacetobacter_diazotrophicus_PA1_5_uid61587 COG3513 cas9 479173968 Gordonibacter_pamelaeae_7_10_1_b_uid197167 mkCas0193 cas9 345430422 Haemophilus_parainfluenzae_T3T1_uid72801 COG3513 cas9 471315929 Helicobacter_cinaedi_ATCC_BAA_847_uid193765 COG3513 cas9 386762035 Helicobacter_cinaedi_PAGU611_uid162219 COG3513 cas9 291276265 Helicobacter_mustelae_12198_uid46647 COG3513 cas9 385811609 Ignavibacterium_album_JCM_16511_uid162097 cd09643, COG3513 cas9 310780384 Ilyobacter_polytropus_DSM_2926_uid59769 COG3513 cas9 331702228 Lactobacillus_buchneri_NRRL_B_30929_uid66205 mkCas0193 cas9 406027703 Lactobacillus_buchneri_uid73657 mkCas0193 cas9 385824065 Lactobacillus_casei_BD_II_uid162119 mkCas0193 cas9 191639137 Lactobacillus_casei_BL23_uid59237 mkCas0193 cas9 385820880 Lactobacillus_casei_LC2W_uid162121 mkCas0193 cas9 523514789 Lactobacillus_casei_LOCK919_uid210959 mkCas0193 cas9 409997999 Lactobacillus_casei_W56_uid178736 mkCas0193 cas9 301067199 Lactobacillus_casei_Zhang_uid50673 mkCas0193 cas9 385815562 Lactobacillus_delbrueckii_bulgaricus_2038_uid161929 mkCas0193 cas9 385815563 Lactobacillus_delbrueckii_bulgaricus_2038_uid161929 mkCas0193 cas9 385815564 Lactobacillus_delbrueckii_bulgaricus_2038_uid161929 mkCas0193 cas9 385826041 Lactobacillus_johnsonii_DPC_6026_uid162057 mkCas0193 cas9 532357525 Lactobacillus_paracasei_8700_2_uid55295 mkCas0193 cas9 448819853 Lactobacillus_plantarum_ZJ316_uid188689 mkCas0193 cas9 385828839 Lactobacillus_rhamnosus_GG_uid161983 mkCas0193 cas9 258509199 Lactobacillus_rhamnosus_GG_uid59313 mkCas0193 cas9 523517690 Lactobacillus_rhamnosus_LOCK900_uid210957 mkCas0193 cas9 385839898 Lactobacillus_salivarius_CECT_5713_uid162005 mkCas0193 cas9 385839899 Lactobacillus_salivarius_CECT_5713_uid162005 mkCas0193 cas9 385839900 Lactobacillus_salivarius_CECT_5713_uid162005 mkCas0193 cas9 90961083 Lactobacillus_salivarius_UCC118_uid58233 mkCas0193 cas9 90961084 Lactobacillus_salivarius_UCC118_uid58233 mkCas0193 cas9 347534532 Lactobacillus_sanfranciscensis_TMW_1_1304_uid72937 mkCas0193 cas9 54296138 Legionella_pneumophila_Paris_uid58211 cd09704 cas9 406600271 Leuconostoc_gelidum_JB7_uid175682 mkCas0193 cas9 16801805 Listeria_innocua_Clip11262_uid61567 cd09643, COG3513 cas9 386044902 Listeria_monocytogenes_10403S_uid54461 COG3513, COG3513 cas9 550898770 Listeria_monocytogenes_EGD_uid223288 COG3513, COG3513 cas9 386048324 Listeria_monocytogenes_J0161_uid54459 COG3513, COG3513 cas9 405756714 Listeria_monocytogenes_SLCC2540_uid175106 COG3513, COG3513 cas9 404411844 Listeria_monocytogenes_SLCC5850_uid175110 COG3513, COG3513 cas9 404282159 Listeria_monocytogenes_serotype_1_2b_SLCC2755_uid52455 COG3513, COG3513 cas9 404287973 Listeria_monocytogenes_serotype_7_SLCC2482_uid174871 COG3513, COG3513 cas9 433625054 Mycoplasma_cynos_C142_uid184824 cd09643 cas9 401771107 Mycoplasma_gallisepticum_CA06_2006_052_5_2P_uid172630 cd09643 cas9 385326554 Mycoplasma_gallisepticum_F_uid162001 cd09643 cas9 401767318 Mycoplasma_gallisepticum_NC95_13295_2_2P_uid172625 cd09643 cas9 401768090 Mycoplasma_gallisepticum_NC96_1596_4_2P_uid172626 cd09643 cas9 401768851 Mycoplasma_gallisepticum_NY01_2001_047_5_1P_uid172627 cd09643 cas9 385325798 Mycoplasma_gallisepticum_R_high_uid161999 cd09643 cas9 294660600 Mycoplasma_gallisepticum_R_low_uid57993 cd09643 cas9 565627373 Mycoplasma_gallisepticum_S6_uid200523 cd09643 cas9 401769598 Mycoplasma_gallisepticum_WI01_2001_043_13_2P_uid172628 cd09643 cas9 47458868 Mycoplasma_mobile_163K_uid58077 cd09643 cas9 71894592 Mycoplasma_synoviae_53_uid58061 cd09643 cas9 313669044 Neisseria_lactamica_020_06_uid60851 COG3513 cas9 161869390 Neisseria_meningitidis_053442_uid58587 COG3513 cas9 385324780 Neisseria_meningitidis_8013_uid161967 COG3513 cas9 385337435 Neisseria_meningitidis_WUE_2594_uid162093 COG3513 cas9 218767588 Neisseria_meningitidis_Z2491_uid57819 COG3513 cas9 254804356 Neisseria_meningitidis_alpha14_uid61649 COG3513 cas9 319957206 Nitratifractor_salsuginis_DSM_16511_uid62183 cd09643 cas9 325983496 Nitrosomonas_AL212_uid55727 COG3513 cas9 302336020 Olsenella_uli_DSM_7084_uid51367 mkCas0193 cas9 392391493 Ornithobacterium_rhinotracheale_DSM_15997_uid168256 cd09643 cas9 154250555 Parvibaculum_lavamentivorans_DS_1_uid58739 cd09643 cas9 15602992 Pasteurella_multocida_Pm70_uid57627 COG3513 cas9 557607382 Pediococcus_pentosaceus_SL4_uid227215 mkCas0193 cas9 294674019 Prevotella_ruminicola_23_uid47507 COG3513 cas9 408489713 Psychroflexus_torquis_ATCC_700755_uid54205 cd09643, cd09643 cas9 90425961 Rhodopseudomonas_palustris_BisB18_uid58443 COG3513 cas9 91975509 Rhodopseudomonas_palustris_BisB5_uid58441 COG3513 cas9 83591793 Rhodospirillum_rubrum_ATCC_11170_uid57655 cd09643 cas9 386348484 Rhodospirillum_rubrum_F11_uid162149 cd09643 cas9 383485594 Riemerella_anatipestifer_ATCC_11845_DSM_15868_uid159857 COG3513, cd09643 cas9 407451859 Riemerella_anatipestifer_RA_CH_1_uid175469 COG3513, cd09643 cas9 442314523 Riemerella_anatipestifer_RA_CH_2_uid186548 COG3513, cd09643 cas9 386321727 Riemerella_anatipestifer_RA_GD_uid162013 COG3513, cd09643 cas9 479204792 Roseburia_intestinalis_uid197164 COG3513 cas9 470213512 Sphingomonas_MM_1_uid193771 COG3513 cas9 325972003 Spirochaeta_Buddy_uid63633 cd09643 cas9 563693590 Spiroplasma_apis_B31_uid230613 cd09643 cas9 507384108 Spiroplasma_syrphidicola_EA_l_uid205054 cd09643 cas9 556591142 Staphylococcus_pasteuri_SP1_uid226267 cd09643 cas9 386318630 Staphylococcus_pseudintermedius_ED99_uid162109 mkCas0193 cas9 269123826 Streptobacillus_moniliformis_DSM_12112_uid41863 COG3513 cas9 552737657 Streptococcus_I_G2_uid224251 cd09643 cas9 512539130 Streptococcus_agalactiae_09mas018883_uid208674 mkCas0193 cas9 22537057 Streptococcus_agalactiae_2603V_R_uid57943 mkCas0193 cas9 494703075 Streptococcus_agalactiae_2_22_uid202215 mkCas0193 cas9 76788458 Streptococcus_agalactiae_A909_uid57935 mkCas0193 cas9 406709383 Streptococcus_agalactiae_GD201008_001_uid175780 mkCas0193 cas9 512544670 Streptococcus_agalactiae_ILRI005_uid208676 mkCas0193 cas9 512698372 Streptococcus_agalactiae_ILRI112_uid208675 mkCas0193 cas9 25010965 Streptococcus_agalactiae_NEM316_uid61585 mkCas0193 cas9 410594450 Streptococcus_agalactiae_SA20_06_uid178722 mkCas0193 cas9 538370328 Streptococcus_anginosus_C1051_uid218003 cd09643 cas9 410494913 Streptococcus_dysgalactiae_equisimilis_AC_2713_uid178644 COG3513 cas9 386317166 Streptococcus_dysgalactiae_equisimilis_ATCC_12394_uid161979 COG3513 cas9 251782637 Streptococcus_dysgalactiae_equisimilis_GGS_124_uid59103 COG3513 cas9 408401787 Streptococcus_dysgalactiae_equisimilis_RE378_uid176684 COG3513 cas9 195978435 Streptococcus_equi_zooepidemicus_MGCS10565_uid59263 COG3513 cas9 386338081 Streptococcus_gallolyticus_ATCC_43143_uid162103 cd09643 cas9 386338091 Streptococcus_gallolyticus_ATCC_43143_uid162103 mkCas0193 cas9 325978669 Streptococcus_gallolyticus_ATCC_BAA_2069_uid63617 mkCas0193 cas9 288905632 Streptococcus_gallolyticus_UCN34_uid46061 cd09643 cas9 288905639 Streptococcus_gallolyticus_UCN34_uid46061 mkCas0193 cas9 157150687 Streptococcus_gordonii_Challis_substr CH1_uid57667 cd09643 cas9 379705580 Streptococcus_infantarius_CJ18_uid87033 mkCas0193 cas9 508127396 Streptococcus_iniae_SF1_uid206041 mkCas0193 cas9 508127399 Streptococcus_iniae_SF1_uid206041 COG3513 cas9 538379999 Streptococcus_intermedius_B196_uid218000 cd09643 cas9 527330434 Streptococcus_lutetiensis_033_uid213397 mkCas0193 cas9 374338350 Streptococcus_macedonicus_ACA_DC_198_uid81631 cd09643 cas9 397650022 Streptococcus_mutans_GS_5_uid169223 mkCas0193 cas9 387785882 Streptococcus_mutans_LJ23_uid162197 mkCas0193 cas9 290580220 Streptococcus_mutans_NN2025_uid46353 mkCas0193 cas9 24379809 Streptococcus_mutans_UA159_uid57947 mkCas0193 cas9 336064611 Streptococcus_pasteurianus_ATCC_43144_uid68019 cd09643 cas9 410680443 Streptococcus_pyogenes_A20_uid178106 COG3513 cas9 470200927 Streptococcus_pyogenes_M1_476_uid193766 COG3513 cas9 15675041 Streptococcus_pyogenes_M1_GAS_uid57845 COG3513 cas9 94990395 Streptococcus_pyogenes_MGAS10270_uid58571 COG3513 cas9 94994317 Streptococcus_pyogenes_MGAS10750_uid58575 COG3513 cas9 383479946 Streptococcus_pyogenes_MGAS15252_uid158037 COG3513 cas9 383493861 Streptococcus_pyogenes_MGAS1882_uid158061 COG3513 cas9 94992340 Streptococcus_pyogenes_MGAS2096_uid58573 COG3513 cas9 21910213 Streptococcus_pyogenes_MGAS315_uid57911 COG3513 cas9 71910582 Streptococcus_pyogenes_MGAS5005_uid58337 COG3513 cas9 71903413 Streptococcus_pyogenes_MGAS6180_uid58335 COG3513 cas9 94988516 Streptococcus_pyogenes_MGAS9429_uid58569 COG3513 cas9 209559356 Streptococcus_pyogenes_NZ131_uid59035 COG3513 cas9 28896088 Streptococcus_pyogenes_SSI_ 1_uid57895 COG3513 cas9 387783792 Streptococcus_salivarius_JIM8777_uid162145 cd09643 cas9 386584496 Streptococcus_suis_D9_uid162125 cd09643 cas9 389856936 Streptococcus_suis_ST1_uid167482 mkCas0193 cas9 330833104 Streptococcus_suis_ST3_uid66327 cd09643 cas9 55822627 Streptococcus_thermophilus_CNRZ1066_uid58221 cd09643 cas9 386344353 Streptococcus_thermophilus_JIM_8232_uid162157 cd09643 cas9 116627542 Streptococcus_thermophilus_LMD_9_uid58327 cd09643 cas9 116628213 Streptococcus_thermophilus_LMD_9_uid58327 mkCas0193 cas9 55820735 Streptococcus_thermophilus_LMG_18311_uid58219 cd09643 cas9 387909441 Streptococcus_thermophilus_MN_ZLW_002_uid166827 cd09643 cas9 387910220 Streptococcus_thermophilus_MN_ZLW_002_uid166827 mkCas0193 cas9 386086348 Streptococcus_thermophilus_ND03_uid162015 cd09643 cas9 386087120 Streptococcus_thermophilus_ND03_uid162015 mkCas0193 cas9 389874754 Tistrella_mobilis_KA081020_065_uid167486 COG3513 cas9 42525843 Treponema_denticola_ATCC_35405_uid57583 mkCas0193 cas9 530892607 Treponema_pedis_T_A4_uid215715 COG3513, COG3513 cas9 121608211 Verminephrobacter_eiseniae_EF01_2_uid58675 cd09643 cas9 525888882 Vibrio_parahaemolyticus_O1_K33_CDC_K4557_uid212977 COG3513, COG3513 cas9 525913263 Vibrio_parahaemolyticus_O1_K33_CDC_K4557_uid212977 COG3513 cas9 525919586 Vibrio_parahaemolyticus_O1_K33_CDC_K4557_uid212977 COG3513, COG3513 cas9 525927253 Vibrio_parahaemolyticus_O1_K33_CDC_K4557_uid212977 COG3513, COG3513 cas9 325955459 Weeksella_virosa_DSM_16922_uid63627 cd09643, cd09643 cas9 34557790 Wolinella_succinogenes_DSM_1740_uid61591 cd09643 cas9 34557932 Wolinella_succinogenes_DSM_1740_uid61591 cd09704 cas9 295136244 Zunongwangia_profunda_SM_A87_uid48073 COG3513, cd09643 cas9 304313029 gamma_proteobacterium_HdN1_uid51635 cd09643 cas9 189485058 uncultured_Termite_group_1_bacterium_phylotype_ cd09643 cas9 Rs_D17_uid59059 189485059 uncultured_Termite_group_l_bacterium_phylotype_ cd09643 cas9 Rs_D17_uid59059 189485225 uncultured_Termite_group_1_bacterium_phylotype_ COG3513 cas9 Rs_D17_uid59059 347536497 Flavobacterium_branchiophilum_FL_15_uid73421 COG3513, cd09643, cas9, cas9 COG3513 365959402 Flavobacterium_columnare_ATCC_49512_uid80731 COG3513, cd09643, cas9, cas9 COG3513 387132277 Prevotella_intermedia_17_uid163151 cd09643, COG3513, cas9, Type COG0188 IIA topoisome rase

Guide RNA (gRNA)

gRNA or sgRNA (single guide RNA) is engineered as a fusion between a crRNA and part of the transactivating CRISPR RNA (tracrRNA) sequence, which guides the Cas9 to a specific target DNA sequence that is complementary to the protospacer region. Guide RNA may include an expression cassette containing a chimeric RNA design with a long tracrRNA hybrid, short tracrRNA hybrid or a native CRISPR array+tracrRNA conformation. Chimeric gRNA combines the targeting specificity of the crRNA with the scaffolding properties of the tracrRNA into a single transcript. gRNA transient expression is controlled by species-specific higher plant RNA Polymerase III promoters such as those from the U6 or U3 snRNA gene family (Wang et al., 2008). gRNA transcript termination is controlled by a 6-20 nucleotide tract of poly dT as per Wang et al., 2008. gRNA expression cassettes are located on the same or different transient expression vectors from other components of the CRISPR-Cas system. gRNA transcripts may be synthesized in vitro and delivered directly into plant cells, independent of or in combination with gRNA transient expression vectors.

In some embodiments, the native S. pyogenes type II CRISPR-Cas system consists of a Crispr ASsociated (Cas9) nuclease and two disparate non-coding RNAs, trans-activating RNA (tracrRNA) and CRISPR RNA (crRNA). The RNA components of this system direct Cas9 nuclease to a sequence specific target in a genome. All three components can be expressed separately as tracrRNA and crRNA and Cas9 protein. The crRNA provides the target specificity and consists of a spacer sequence of 20 bases that are complementary to the target DNA (protospacer sequence) that is cleaved by Cas9 (Le Cong et al., 2013). The tracrRNA acts as an RNA scaffold when associated with crRNA by way of RNA:RNA base pairing and it is this complex that associates with Cas9. The tracrRNA can be engineered to be shorter than 89 bases, as is the case in the Alt-R™ system developed by Integrated DNA Technologies (IDT). In this system tracrRNA as short as 67 bases have increased on-target performance when compare to native tracrRNA. When the crRNA and tracrRNA are artificially combined into a single fused functional RNA or single guide RNA (sgRNA) targeting of Cas9 protein can be greatly simplified over the native system. Similar to the native tracerRNA:crRNA complex, the engineered sgRNA guides the Cas9 to a specific target DNA sequence.

Target Region

Guide RNAs contain two components that define specificity to a DNA target region, a proto-spacer and a proto-spacer adjacent motif (PAM). Proto-spacer sequence, typically 20 nucleotides but can vary based on the DNA target, provides DNA sequence specificity for the CRISPR-Cas complex. DNA targets also contain a NNG or NAG tri-nucleotide sequence (PAM) where N denotes any nucleotide, immediately 3′ or downstream of the proto-spacer.

One Component Approach

Similar to Le Cong et al. (2013) and others, a simplified “one component approach” to CRISPR-Cas gene editing wherein a single transient expression construct contains all components of the CRISPR-Cas complex, i.e. both the gRNA and the Cas expressions cassettes. This allows for an easy modular design for targeting single or multiple loci in any given plant or crop. Targeting multiple loci can be achieved by simply swapping in the target-specific gRNA cassettes. Additionally, species specific promoters, terminators or other expressing enhancing elements can easily be shuttled in and out of “one component approach” transient vectors allowing for optimal expression of both gRNA and Cas protein in a species-specific manner.

Two Component Approach

In the two-component approach, Cas and gRNA expression cassettes are located on different transient expression vectors. This allows for delivery of a CRISPR-Cas editing components separately, allowing for different ratios of gRNA to Cas within the same cell. Similar to the one component approach, the two-component approach also allows for promoters, terminators or other elements affecting expression of CRISPR-Cas components to be easily altered and allow targeting of DNA in a species-specific manner.

Antibiotics

Another class of endonucleases are antibiotics which are DNA cleaving glycopeptides such as the bleomycin family of antibiotics are DNA cleaving glycopeptides which include bleomycin, zeocin, phleomycin, tallysomycin, pepleomycin and others which are further described herein.

Other DNA-Modifying Molecules

Other DNA-modifying molecules may be used in targeted gene recombination. For example, peptide nucleic acids may be used to induce modifications to the genome of the target cell or cells (see, e.g., Ecker, U.S. Pat. No. 5,986,053 herein incorporated by reference). In brief, synthetic nucleotides comprising, at least, a partial peptide backbone is used to target a homologous genomic nucleotide sequence. Upon binding to the double-helical DNA, or through a mutagen ligated to the peptide nucleic acid, modification of the target DNA sequence and/or recombination is induced to take place. Targeting specificity is determined by the degree of sequence homology between the targeting sequence and the genomic sequence.

In some embodiments of the methods and compositions of the present disclosure genes (such as the SHP gene) may be targeted using triple helix forming oligonucleotides (TFO). TFOs may be generated synthetically, for example, by PCR or by use of a gene synthesizer apparatus. Additionally, TFOs may be isolated from genomic DNA if suitable natural sequences are found. TFOs may be used in a number of ways, including, for example, by tethering to a mutagen such as, but not limited to, psoralen or chlorambucil (see, e.g., Havre et al., Proc Nat'l Acad Sci, U.S.A. 90:7879-7883, 1993; Havre et al., J Virol 67:7323-7331, 1993; Wang et al., Mol Cell Biol 15:1759-1768, 1995; Takasugi et al., Proc Nat'l Acad Sci, U.S.A. 88:5602-5606, 1991; Belousov et al., Nucleic Acids Res 25:3440-3444, 1997). Furthermore, for example, TFOs may be tethered to donor duplex DNA (see, e.g., Chan et al., J Biol Chem 272:11541-11548, 1999). TFOs can also act by binding with sufficient affinity to provoke error-prone repair (Wang et al., Science 271:802-805, 1996).

The methods disclosed herein are not necessarily limited to the nature or type of DNA-modifying reagent which is used. For example, such DNA-modifying reagents release radicals which result in DNA strand breakage. Alternatively, the reagents alkylate DNA to form adducts which would block replication and transcription. In another alternative, the reagents generate crosslinks or molecules that inhibit cellular enzymes leading to strand breaks. Examples of DNA-modifying reagents which have been linked to oligonucleotides to form TFOs include, but are not limited to, indolocarbazoles, napthalene diimide (NDI), transplatin, bleomycin, analogues of cyclopropapyrroloindole, and phenanthodihydrodioxins. In particular, indolocarbazoles are topoisomerase I inhibitors. Inhibition of these enzymes results in strand breaks and DNA protein adduct formation (Arimondo et al., Bioorganic and Medicinal Chem. 8, 777-784, 2000). NDI is a photooxidant that can oxidize guanines which could cause mutations at sites of guanine residues (Nunez et al., Biochemistry, 39, 6190-6199, 2000). Transplatin has been shown to react with DNA in a triplex target when the TFO is linked to the reagent. This reaction causes the formation of DNA adducts which would be mutagenic (Columbier et al., Nucleic Acids Research, 24: 4519-4524, 1996). Bleomycin is a DNA breaker, widely used as a radiation mimetic. It has been linked to oligonucleotides and shown to be active as a breaker in that format (Sergeyev, Nucleic Acids Research 23, 4400-4406, 1995; Kane et al., Biochemistry, 34, 16715-16724, 1995). Analogues of cyclopropapyrroloindole have been linked to TFOs and shown to alkylate DNA in a triplex target sequence. The alkylated DNA would then contain chemical adducts which would be mutagenic (Lukhtanov et al., Nucleic Acids Research, 25, 5077-5084, 1997). Phenanthodihydrodioxins are masked quinones that release radical species upon photoactivation. They have been linked to TFOs and have been shown to introduce breaks into duplex DNA on photoactivation (Bendinskas et al., Bioconjugate Chem. 9, 555-563, 1998).

Other methods of inducing modifications and/or recombination are contemplated by the present disclosure. For example, another embodiment involves the induction of homologous recombination between an exogenous DNA fragment and the targeted gene (see e.g., Capecchi et al., Science 244:1288-1292, 1989) or by using peptide nucleic acids (PNA) with affinity for the targeted site. Still other methods include sequence specific DNA recognition and targeting by polyamides (see e.g., Dervan et al., Curr Opin Chem Biol 3:688-693, 1999; Biochemistry 38:2143-2151, 1999) and the use nucleases with site specific activity (e.g., zinc finger proteins, TALENs, Meganucleases and/or CRISPRs).

The present disclosure is not limited to any particular frequency of modification and/or recombination. In some embodiments the methods disclosed herein result in a frequency of modification in the target nucleotide sequence of from 0.01% to 3%. Nonetheless, any frequency (i.e., between 0% and 100%) of modification and/or recombination is contemplated to be within the scope of the present disclosure. The frequency of modification and/or recombination is dependent on the method used to induce the modification and/or recombination, the cell type used, the specific gene targeted, and the DNA mutating reagent used, if any. Additionally, the method used to detect the modification and/or recombination, due to limitations in the detection method, may not detect all occurrences of modification and/or recombination. Furthermore, some modification and/or recombination events may be silent, giving no detectable indication that the modification and/or recombination has taken place. The inability to detect silent modification and/or recombination events gives an artificially low estimate of modification and/or recombination. Because of these reasons, and others, the disclosure is not necessarily limited to any particular modification and/or recombination frequency. In one embodiment, the frequency of modification and/or recombination is between 0.01% and 100%. In another embodiment, the frequency of modification and/or recombination is between 0.01% and 50%. In yet another embodiment, the frequency of modification and/or recombination is between 0.1% and 10%. In still yet another embodiment, the frequency of modification and/or recombination is between 0.1% and 5%.

The term “frequency of mutation” as used herein in reference to a population of cells which are treated with a DNA-modifying molecule that is capable of introducing a mutation into a target site in the cells' genome, refers to the number of cells in the treated population which contain the mutation at the target site as compared to the total number of cells which are treated with the DNA-modifying molecule. For example, with respect to a population of cells which is treated with the DNA-modifying molecule TFO tethered to psoralen which is designed to introduce a mutation at a target site in the cells' genome, a frequency of mutation of 5% means that of a total of 100 cells which are treated with TFO-psoralen, 5 cells contain a mutation at the target site.

Although the present disclosure is not necessarily limited to any degree of precision in the modification and/or recombination of DNA in the cell, it is contemplated that some embodiments of the present disclosure require higher degrees of precision, depending on the desired result. For example, the specific sequence changes required for gene repair (e.g., particular base changes) require a higher degree of precision as compared to producing a gene knockout wherein only the disruption of the gene is necessary. With the methods of the present disclosure, achievement of higher levels of precision in modification and/or homologous recombination techniques is greater than with prior art methods.

Delivery of Gene Repair Oligonucleobases into Plant Cells

Any commonly known method used to transform a plant cell can be used for delivering the gene repair oligonucleobases. Illustrative methods are listed below. The methods and compositions herein may involve any of many methods to transfect the cells with the DNA-modifying reagent or reagents. Methods for the introduction of DNA modifying reagents into a cell or cells are well known in the art and include, but are not limited to, microinjection, electroporation, passive adsorption, calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, liposome fusion, lipofectin, nucleofection, protoplast fusion, retroviral infection, biolistics (i.e., particle bombardment) and the like.

The use of metallic microcarriers (microspheres) for introducing large fragments of DNA into plant cells having cellulose cell walls by projectile penetration is well known to those skilled in the relevant art (henceforth biolistic delivery). U.S. Pat. Nos. 4,945,050; 5,100,792 and 5,204,253 describe general techniques for selecting microcarriers and devices for projecting them.

Specific conditions for using microcarriers in the methods disclosed herein may include the conditions described in International Publication WO 99/07865. In an illustrative technique, ice cold microcarriers (60 mg/mL), mixed duplex oligonucleotide (60 mg/mL) 2.5 M CaCl₂) and 0.1 M spermidine are added in that order; the mixture gently agitated, e.g., by vortexing, for 10 minutes and then left at room temperature for 10 minutes, whereupon the microcarriers are diluted in 5 volumes of ethanol, centrifuged and resuspended in 100% ethanol. Good results can be obtained with a concentration in the adhering solution of 8-10 μg/μL microcarriers, 14-17 μg/mL mixed duplex oligonucleotide, 1.1-1.4 M CaCl₂) and 18-22 mM spermidine. Optimal results were observed under the conditions of 8 μg/μL microcarriers, 16.5 μg/mL mixed duplex oligonucleotide, 1.3 M CaCl₂) and 21 mM spermidine.

Gene repair oligonucleobases can also be introduced into plant cells using microfibers to penetrate the cell wall and cell membrane. U.S. Pat. No. 5,302,523 to Coffee, R., and Dunwell, J. M. (1994) describes the use of silicon carbide fibers to facilitate transformation of suspension maize cultures of Black Mexican Sweet. Any mechanical technique that can be used to introduce DNA for transformation of a plant cell using microfibers can be used to deliver gene repair oligonucleobases for transmutation.

An illustrative technique for microfiber delivery of a gene repair oligonucleobase is as follows: Sterile microfibers (2 μg) are suspended in 150 μL of plant culture medium containing about 10 μg of a mixed duplex oligonucleotide. A suspension culture is allowed to settle, and equal volumes of packed cells and the sterile fiber/nucleotide suspension are vortexed for 10 minutes and plated. Selective media are applied immediately or with a delay of up to about 120 hours as is appropriate for the particular trait.

In an alternative embodiment, the gene repair oligonucleobases can be delivered to the plant cell by electroporation of a protoplast derived from a plant part. The protoplasts are formed by enzymatic treatment of a plant part, particularly a leaf, according to techniques well known to those skilled in the art. See, e.g., Gallois et al., 1996, in Methods in Molecular Biology 55:89-107, Humana Press, Totowa, N.J.; Kipp et al., 1999, in Methods in Molecular Biology 133:213-221, Humana Press, Totowa, N.J. The protoplasts need not be cultured in growth media prior to electroporation. Illustrative conditions for electroporation are 300,000 protoplasts in a total volume of 0.3 mL with a concentration of gene repair oligonucleobase of between 0.6-4 μg/mL.

In an alternative embodiment, nucleic acids are taken up by plant protoplasts in the presence of the membrane-modifying agent polyethylene glycol, according to techniques well known to those skilled in the art. In another alternative embodiment, the gene repair oligonucleobases can be delivered by injecting it with a microcapillary into plant cells or into protoplasts.

In an alternative embodiment, nucleic acids are embedded in microbeads composed of calcium alginate and taken up by plant protoplasts in the presence of the membrane-modifying agent polyethylene glycol (see, e.g., Sone et al., Journal of Bioscience and Bioengineering, 94(1):87-91, 2002; Liu et al., 2004).

In an alternative embodiment, nucleic acids frozen in water and introduced into plant cells by bombardment in the form of microparticles (see, e.g., Gilmore, 1991, U.S. Pat. No. 5,219,746; Brinegar et al.).

In an alternative embodiment, nucleic acids attached to nanoparticles are introduced into intact plant cells by incubation of the cells in a suspension containing the nanoparticle (see, e.g., Pasupathy et al., Biotechnology Journal: Healthcare Nutrition Technology, 3(8), 1078-1082, 2008) or by delivering them into intact cells through particle bombardment or into protoplasts by co-incubation (see, e.g., Torney et al., Nature nanotechnology, 2(5), 295, 2007).

In an alternative embodiment, nucleic acids complexed with penetrating peptides and delivered into cells by co-incubation (see, e.g., Chugh and Eudes, Journal of peptide science: an official publication of the European Peptide Society, 14(4), 477-481, 2008; WO 2008148223 A1).

In an alternative embodiment, nucleic acids are introduced into intact cells through electroporation (see, e.g., He et al., 1998, US 2003/0115641 A1, Dobres et al.).

In an alternative embodiment, nucleic acids are delivered into cells of dry embryos by soaking them in a solution with nucleic acids (see, e.g., Töpfer et al., 1989, Senaratna et al., 1991) or in other embodiments are introduced by Cellsqueeze (SQZ Biotech).

Methods of Reducing Polypeptide Activity and Other Mutagenesis Techniques

Certain aspects of the present disclosure relate to reducing levels and/or activity of a polypeptide (e.g. an SHP polypeptide). Methods of modifying decreasing the quantity/level or the activity of one or more polypeptides of the present disclosure are well-known in the art and are described herein.

Cells (e.g. plant cells) of the present disclosure may contain one or more polypeptides with decreased activity as compared to a corresponding control cell, such as a wild-type cell. In some embodiments, one or more SHP proteins have decreased activity in a host cell as compared to a corresponding control cell. Methods of decreasing the expression, abundance, and/or activity of a polypeptide are well-known in the art and are described herein.

In some embodiments, decreasing the activity of a polypeptide such as, for example, one or more SHP proteins involves decreasing the expression of a nucleic acid encoding the polypeptide.

Decreasing the expression of a nucleic acid may be accomplished by introducing a genetic mutation into a target nucleic acid. Mutagenesis approaches may be used to disrupt or “knockout” the expression of a target gene by generating mutations. In some embodiments, the mutagenesis results in a partial deletion of the target gene. In other embodiments, the mutagenesis results in a complete deletion of the target gene. Methods of mutagenizing microorganisms are well known in the art and include, for example, random mutagenesis and site-directed mutagenesis to induce mutations. Examples of methods of random mutagenesis include, for example, chemical mutagenesis (e.g., using ethane methyl sulfonate), insertional mutagenesis, and irradiation. In some embodiments, mutagenic techniques may be used to introduce a premature stop codon into a nucleic acid of the present disclosure (e.g. an SHP gene). This could be accomplished via, for example, a targeted single nucleotide change into the target nucleic acid at a location that creates a premature stop codon.

In some embodiments, nucleic acids of the present disclosure (e.g. SHP genes) may be edited in a manner that does not result in a shift of the open reading frame or that does not substantially eliminate expression of the nucleic acid and/or the polypeptide it encodes, such as, for example introducing a single nucleotide change into the nucleic acid. Various techniques may be used to accomplish such an edit such as, for example, targeted introduction of a point mutation in the nucleic acid. Such edits may, for example, reduce the expression of the nucleic acid and/or reduce the expression and/or activity of the polypeptide it encodes. For example, a point mutation may be introduced into an SHP gene that results in an amino acid change in the encoded polypeptide sequence. In some embodiments, such an amino acid change may be in a region important for the function of the SHP gene, such that the encoded mutant SHP polypeptide has reduced activity and/or altered function.

One method for reducing or inhibiting the expression of a target gene is by genetically modifying the target gene and introducing it into the genome of a host cell to replace the wild-type version of the gene by homologous recombination (for example, as described in U.S. Pat. No. 6,924,146).

Another method for reducing or inhibiting the expression of a target gene is by insertion mutagenesis using the T-DNA of Agrobacterium tumefaciens, or transposons (see Winkler et al., Methods Mol. Biol. 82:129-136, 1989, and Martienssen Proc. Natl. Acad. Sci. 95:2021-2026, 1998). After generating the insertion mutants, the mutants can be screened to identify those containing the insertion in a target gene. Methods to disrupt a target gene by insertional mutagenesis are described in for example, U.S. Pat. No. 5,792,633. Methods to disrupt a target gene by transposon mutagenesis are described in for example, U.S. Pat. No. 6,207,384.

A further method to disrupt a target gene is by use of the cre-lox system (for example, as described in U.S. Pat. No. 4,959,317).

Another method to disrupt a target gene is by use of PCR mutagenesis (for example, as described in U.S. Pat. No. 7,501,275).

Endogenous gene expression may also be reduced or inhibited by means of RNA interference (RNAi), which uses a double-stranded RNA having a sequence identical or similar to the sequence of the target gene. RNAi may include the use of micro RNA, such as artificial miRNA, to suppress expression of a gene.

RNAi is the phenomenon in which when a double-stranded RNA having a sequence identical or similar to that of the target gene is introduced into a cell, the expressions of both the inserted exogenous gene and target endogenous gene are suppressed. The double-stranded RNA may be formed from two separate complementary RNAs or may be a single RNA with internally complementary sequences that form a double-stranded RNA.

Thus, in some embodiments, reduction or inhibition of gene expression is achieved using RNAi techniques. For example, to achieve reduction or inhibition of the expression of a DNA encoding a protein using RNAi, a double-stranded RNA having the sequence of a DNA encoding the protein, or a substantially similar sequence thereof (including those engineered not to translate the protein) or fragment thereof, is introduced into a host cell of interest. As used herein, RNAi and dsRNA both refer to gene-specific silencing that is induced by the introduction of a double-stranded RNA molecule, see e.g., U.S. Pat. Nos. 6,506,559 and 6,573,099, and includes reference to a molecule that has a region that is double-stranded, e.g., a short hairpin RNA molecule. The resulting cells may then be screened for a phenotype associated with the reduced expression of the target gene, e.g., reduced cellulase expression, and/or by monitoring steady-state RNA levels for transcripts of the target gene. Although the sequences used for RNAi need not be completely identical to the target gene, they may be at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the target gene sequence. See, e.g., U.S. Patent Application Publication No. 2004/0029283. The constructs encoding an RNA molecule with a stem-loop structure that is unrelated to the target gene and that is positioned distally to a sequence specific for the gene of interest may also be used to inhibit target gene expression. See, e.g., U.S. Patent Application Publication No. 2003/0221211.

The RNAi nucleic acids may encompass the full-length target RNA or may correspond to a fragment of the target RNA. In some cases, the fragment will have fewer than 100, 200, 300, 400, or 500 nucleotides corresponding to the target sequence. In addition, in some aspects, these fragments are at least, e.g., 50, 100, 150, 200, or more nucleotides in length. Interfering RNAs may be designed based on short duplexes (i.e., short regions of double-stranded sequences). Typically, the short duplex is at least about 15, 20, or 25-50 nucleotides in length (e.g., each complementary sequence of the double stranded RNA is 15-50 nucleotides in length), often about 20-30 nucleotides, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some cases, fragments for use in RNAi will correspond to regions of a target protein that do not occur in other proteins in the organism or that have little similarity to other transcripts in the organism, e.g., selected by comparison to sequences in analyzing publicly-available sequence databases. Similarly, RNAi fragments may be selected for similarity or identity with a conserved sequence of a gene family of interest, such as those described herein, so that the RNAi targets multiple different gene transcripts containing the conserved sequence.

RNAi may be introduced into a host cell as part of a larger DNA construct. Often, such constructs allow stable expression of the RNAi in cells after introduction, e.g., by integration of the construct into the host genome. Thus, expression vectors that continually express RNAi in cells transfected with the vectors may be employed for this disclosure. For example, vectors that express small hairpin or stem-loop structure RNAs, or precursors to microRNA, which get processed in vivo into small RNAi molecules capable of carrying out gene-specific silencing (Brummelkamp et al, Science 296:550-553, (2002); and Paddison, et al., Genes & Dev. 16:948-958, (2002)) can be used. Post-transcriptional gene silencing by double-stranded RNA is discussed in further detail by Hammond et al., Nature Rev Gen 2: 110-119, (2001); Fire et al., Nature 391: 806-811, (1998); and Timmons and Fire, Nature 395: 854, (1998).

Methods for selection and design of sequences that generate RNAi are well-known in the art (e.g. U.S. Pat. Nos. 6,506,559; 6,511,824; and 6,489,127).

A reduction or inhibition of gene expression in a host cell of a target gene may also be obtained by introducing into host cells antisense constructs based on a target gene nucleic acid sequence. For antisense suppression, a target sequence is arranged in reverse orientation relative to the promoter sequence in the expression vector. The introduced sequence need not be a full length cDNA or gene, and need not be identical to the target cDNA or a gene found in the cell to be transformed. Generally, however, where the introduced sequence is of shorter length, a higher degree of homology to the native target sequence is used to achieve effective antisense suppression. In some aspects, the introduced antisense sequence in the vector will be at least 30 nucleotides in length, and improved antisense suppression will typically be observed as the length of the antisense sequence increases. In some aspects, the length of the antisense sequence in the vector will be greater than 100 nucleotides. Transcription of an antisense construct as described results in the production of RNA molecules that are the reverse complement of mRNA molecules transcribed from an endogenous target gene. Suppression of a target gene expression can also be achieved using a ribozyme. The production and use of ribozymes are disclosed in U.S. Pat. Nos. 4,987,071 and 5,543,508.

Expression cassettes containing nucleic acids that encode target gene expression inhibitors, e.g., an antisense or siRNA, can be constructed using methods well known in the art. Constructs include regulatory elements, including promoters and other sequences for expression and selection of cells that express the construct. Typically, fungal and/or bacterial transformation vectors include one or more cloned coding sequences (genomic or cDNA) under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such transformation vectors typically also contain a promoter (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated expression), a transcription initiation start site, an RNA processing signal (such as intron splice sites), a transcription termination site, and/or a polyadenylation signal.

In certain embodiments, a portion of the target nucleic acid may be modified, such as the region encoding the catalytic domain, the coding region, or a control sequence required for expression of the coding region. Such a control sequence of the gene may be a promoter sequence or a functional part thereof, i.e., a part that is sufficient for affecting expression of the gene. For example, a promoter sequence may be inactivated resulting in no expression or a weaker promoter may be substituted for the native promoter sequence to reduce expression of the coding sequence. Other control sequences for possible modification may include, for example, a leader sequence, a propeptide sequence, a signal sequence, a transcription terminator, and a transcriptional activator.

Plants of the Present Disclosure

The methods and compositions described herein may in certain aspects and embodiments be applicable to plants generally. For example, in some aspects and/or embodiments a plant species may be selected from the Brassicaceae family, including a number of important crops such as Brassica napus (canola, oilseed rape), Brassica rapa (e.g., turnip, Chinese cabbage), Brassica oleracea (broccoli, cabbage, cauliflower, etc.), Brassica juncea (mustard), or Raphanus sativus (common radish), as well as many important legume crops such as peas, beans, lentils, and soybeans.

According to the present description, substantially normal growth of a plant, plant organ, plant tissue or plant cell is defined as a growth rate or rate of cell division of the plant, plant organ, plant tissue, or plant cell that is at least 35%, at least 50%, at least 60%, or at least 75% of the growth rate or rate of cell division in a corresponding plant, plant organ, plant tissue or plant cell expressing the wild type SHP protein.

According to the present description, substantially normal development of a plant, plant organ, plant tissue or plant cell is defined as the occurrence of one or more developmental events in the plant, plant organ, plant tissue or plant cell that are substantially the same as those occurring in a corresponding plant, plant organ, plant tissue or plant cell expressing the wild type SHP protein.

According to the present description plant organs include, but are not limited to, leaves, stems, roots, vegetative buds, floral buds, meristems, embryos, cotyledons, endosperm, sepals, petals, pistils, carpels, stamens, anthers, microspores, pollen, pollen tubes, ovules, ovaries and fruits, or sections, slices or discs taken therefrom. Plant tissues include, but are not limited to, callus tissues, ground tissues, vascular tissues, storage tissues, meristematic tissues, leaf tissues, shoot tissues, root tissues, gall tissues, plant tumor tissues, and reproductive tissues. Plant cells include, but are not limited to, isolated cells with cell walls, variously sized aggregates thereof, and protoplasts.

Plants of the present disclosure include those plants that have the potential to exhibit pod shatter. For example, the present disclosure includes Brassica spp. plants that exhibit pod shatter.

In various embodiments, plants as disclosed herein are principally focused on monocotyledonous plants including any woody plant species that grows as a tree or shrub, any herbaceous species, or any species that produces edible fruits, seeds or vegetables, or any species that produces colorful or aromatic flowers. For example, the plant maybe selected from a species of plant from the group consisting of canola, sunflower, corn, tobacco, sugar beet, cotton, maize, wheat, barley, rice, alfalfa, barley, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, cassava, potato, carrot, lettuce, onion, soy bean, soya spp, sugar cane, pea, chickpea, field pea, fava bean, lentils, turnip, rutabaga, brussel sprouts, lupin, cauliflower, kale, field beans, poplar, pine, eucalyptus, grape, citrus, triticale, alfalfa, rye, oats, turf and forage grasses, flax, oilseed rape, mustard, cucumber, morning glory, balsam, pepper, eggplant, marigold, lotus, cabbage, daisy, carnation, tulip, iris, lily, and nut producing plants insofar as they are not already specifically mentioned.

Plants and plant cells can be tested for resistance preharvest dehiscence using commonly known methods in the art.

In some embodiments, plants of the present disclosure have one or more mutations in one or more SHP genes have increased resistance/reduced susceptibility to preharvest dehiscence as compared to a corresponding control plant (e.g. a plant of the same species that does not have any mutations in any SHP genes, such as a wild-type plant). The incidence of pod shattering in plants having increased resistance/reduced susceptibility to preharvest dehiscence may be, for example, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% lower or reduced as compared to a corresponding control.

As used herein, substantially normal growth of a plant, plant organ, plant tissue or plant cell is defined as a growth rate or rate of cell division of the plant, plant organ, plant tissue, or plant cell that is at least 35%, at least 50%, at least 60%, or at least 75% of the growth rate or rate of cell division in a corresponding plant, plant organ, plant tissue or plant cell expressing the wild-type protein of interest.

As used herein, substantially normal development of a plant, plant organ, plant tissue or plant cell is defined as the occurrence of one or more development events in the plant, plant organ, plant tissue or plant cell that are substantially the same as those occurring in a corresponding plant, plant organ, plant tissue or plant cell expressing the wild-type protein.

In certain embodiments plant organs provided herein include, but are not limited to, leaves, stems, roots, vegetative buds, floral buds, meristems, embryos, cotyledons, endosperm, sepals, petals, pistils, carpels, stamens, anthers, microspores, pollen, pollen tubes, ovules, ovaries and fruits, or sections, slices or discs taken therefrom. Plant tissues include, but are not limited to, callus tissues, ground tissues, vascular tissues, storage tissues, meristematic tissues, leaf tissues, shoot tissues, root tissues, gall tissues, plant tumor tissues, and reproductive tissues. Plant cells include, but are not limited to, isolated cells with cell walls, variously sized aggregates thereof, and protoplasts.

Generation of Plants

Tissue culture of various tissues of plant species and regeneration of plants therefrom is known. For example, the propagation of a canola cultivar by tissue culture is described in any of the following but not limited to any of the following: Li et al., “Somatic embryogenesis in quite a direct way in cultures of mesophyll protoplasts of Brassica napus L.”, Plant Cell Reports 1: 209-211, 1982; Chuong et al., “A Simple Culture Method for Brassica hypocotyls Protoplasts,” Plant Cell Reports 4:4-6, 1985; Barsby et al., “A Rapid and Efficient Alternative Procedure for the Regeneration of Plants from Hypocotyl Protoplasts of Brassica napus,” Plant Cell Reports (Spring, 1996); Kartha et al., “In vitro Plant Formation from Stem Explants of Rape,” Physiol. Plant, 31:217-220, 1974; Narasimhulu et al., “Species Specific Shoot Regeneration Response of Cotyledonary Explants of Brassicas,” Plant Cell Reports (Spring 1988); Sun et al., “Cotyledon-derived diploid and haploid protoplast culture and diploid plant regeneration in Brassica napus cv. ‘Topas’,” Can. J. Bot. 76: 530-541, 1998; Swanson, E., “Microspore Culture in Brassica,” Methods in Molecular Biology, Vol. 6, Chapter 17, p. 159, 1990.

Further reproduction of the variety can occur by tissue culture and regeneration. Tissue culture of various tissues of soybeans and regeneration of plants therefrom is well known and widely published. For example, see Komatsuda et al., “Genotype X Sucrose Interactions for Somatic Embryogenesis in Soybeans,” Crop Sci. 31:333-337, 1991; Stephens et al., “Agronomic Evaluation of Tissue-Culture-Derived Soybean Plants,” Theor. Appl. Genet. 82:633-635, 1991; Komatsuda et al., “Maturation and Germination of Somatic Embryos as Affected by Sucrose and Plant Growth Regulators in Soybeans Glycine gracilis L. Skvortz and Glycine max L. Merr.” Plant Cell, Tissue and Organ Culture, 28:103-113, 1992; Dhir et al., “Regeneration of Fertile Plants from Protoplasts of Soybean (Glycine max L. Merr.); Genotypic Differences in Culture Response,” Plant Cell Reports 11:285-289, 1992; Pandey et al., “Plant Regeneration from Leaf and Hypocotyl Explants of Glycine wightii (W. and A.) VERDC. var. longicauda,” Japan J. Breed. 42:1-5, 1992; and Shetty et al., “Stimulation of In Vitro Shoot Organogenesis in Glycine max L. Merrill. by Allantoin and Amides,” Plant Science 81:245-251, 1992. The disclosures of U.S. Pat. No. 5,024,944 issued Jun. 18, 1991 to Collins et al., and U.S. Pat. No. 5,008,200 issued Apr. 16, 1991 to Ranch et al., are hereby incorporated herein in their entirety by reference.

Certain aspects of the present disclosure also related to plants derived from plants having one or more mutations in a nucleic acid (e.g. an SHP gene) of the present disclosure. For example, plants having one or more SHP mutations may be crossed with the same or different plants to give rise to an F₁ progeny plant, where at least one of the parents of the F₁ progeny plant had the one or more SHP mutations. These F₁ plants can be further self-crossed or crossed with a different plant line, and resulting F₂ progeny can be screened for one or more SHP mutations.

EXAMPLES

The following examples are provided to further illustrate aspects of the present disclosure. These examples are non-limiting and should not be construed as limiting any aspect of the present disclosure.

Example 1: Identification and Characterization of SHP1A, SHP1C, SHP2A, SHP2C, SHP3A, SHP3C, SHP4A and SHP4C Genes

Molecular Characterization of BnSHP Genes

Eight BnSHP genes found in the Brassica genome database (Genoscope) were characterized. The SHP canola genes are herein designated BnSHP1A, BnSHP1C, BnSHP2A, BnSHP2C, BnSHP3A, BnSHP3C, BnSHP4A and BnSHP4C. It appears that each half of the genes were contributed by the B. rapa (AA) and the B. oleracea (CC) parental subgenomes. The respective chromosomal location of each gene in B. napus genome is presented in Table 1, as well as the nucleotide sequence of the coding regions of all eight SHP genes found in Genoscope (SEQ ID NO: 1-8, see Table 1) and the genomic DNA sequence of all eight SHP genes found in Genoscope (SEQ ID NO: 9-16, see Table 1). The partial sequence of the 5′ UTR promoter region, along with some of the 5′coding sequence of each BnSHP gene was cloned and sequenced from genomic DNA obtained from the BN2-SU plant line. All 8 genes were found to have unique promoters. The 5′-UTR of the BnSHP1A gene revealed an insertion of ˜5 kb that appeared to be a translocated transposable element, which is not present in the reference sequence in Genoscope, and may be line-specific. FIG. 1A shows the alignment of the partial nucleotide sequences, while FIG. 1B shows the corresponding translated amino acid sequences obtained from the gene characterization of the 5′ genomic region of the BN2-SU SHP genes, and compared to the corresponding Arabidopsis AtSHP1 and AtSHP2 sequences. The level of homology among all BnSHP genes and the AtSHP genes at the nucleotide and the amino acid levels is very high (>80%).

TABLE 1 Shatterproof genes, chromosomal location (source: Genoscope), source genome, nucleotide coding sequence, and genomic DNA sequence Coding Genomic DNA SHP Gene Position Sequence, Sequence, Cibus Name Chromosome Chromosome Genome SEQ ID NO: SEQ ID NO: BnSHP-1A A09 3139589-3143645 A 1  9 BnSHP-1C C08 29697543-29700152 C 2 10 BnSHP-2A A07 14877625-14881448 A 3 11 BnSHP-2C C06 19706214-19709351 C 4 12 BnSHP-3A A04 1164261-1165954 A 5 13 BnSHP-3C C04 24456162-24474692 C 6 14 BnSHP-4A A05 1619288-1623119 A 7 15 BnSHP-4C Unknown Unknown C 8 16

Cloning and Further Molecular Characterization of BnSHP Genes

SHP1 and 2 genes in Arabidopsis thaliana are highly homologous to the Canola SHP genes having 80% nucleotide identity. Using the publicly available cDNA and genomic sequences of Arabidopsis thaliana SHP1 and SHP2 and those for Brassica napus, PCR primers were designed and used to amplify the BnSHP gene sequences from genomic DNA from elite canola lines BN2 and BN-17. PCR-amplified SHP genomic fragments were cloned and sequenced. Additional sequencing of the genomic DNA fragments was performed by Next Generation Sequencing to complete this analysis. Forward and reverse primers unique to each of the eight SHP genes were used to PCR amplify a fragment from the 5′ UTR through to the region of the SHP genes that encodes for the MADS box domain. Genomic DNA isolated from the haploid B. napus line BN2-SU was used to PCR amplify each gene fragment. The products were cloned into a TOPO TA cloning vector, transformed into competent bacterial cells and plated on LB plates. A minimum of ten colonies for each gene was cloned and sequenced and the resulting sequence was compared to the reference sequences located in GenBank and Genoscope.

Gene Expression Analysis

Canola pods from 6 stages of development were harvested (FIG. 2A). Total RNA was isolated from each sample using gene specific RT-PCR primers to amplify the expressed SHP genes/alleles from cDNA synthesized from total RNA extractions. The cDNA of each sample was then sequenced by NGS to determine the relative expression of each SHP gene at each developmental stage tested.

The expression of all 8 BnSHP genes was investigated to gain insight into which genes are important for the pod shatter reduction phenotype. Expression analysis using gene specific primers resulted in at least 6 of the 8 BnSHP genes expressed in developing siliques (FIG. 2A and FIG. 2B). Sequences corresponding to genes BnSHP1A and BnSHP4C were not represented in these data.

REFERENCES

-   Roeder, A. H. K. and Yanofsky, M. F. (2006) Fruit Development in     Arabidopsis. In, The Arabidopsis Book, American Society of Plant     Biologists, doi: e0075. 10.1199/tab.0075 -   Liljegren, S. J., Ditta, G. S., Eshed, Y., Savidge, B., Bowman, J.     L., and Yanofsky, M. F. (2000) SHATTERPROOF MADS-box genes control     seed dispersal in Arabidopsis. Nature 404, 776-770. -   Raman H, Raman R, Kilian A, Detering F, Carling J, et al. (2014)     Genome-Wide Delineation of Natural Variation for Pod Shatter     Resistance in Brassica napus. PLoS ONE 9(7): e101673.     doi:10.1371/journal.pone.0101673. -   Gururaj K. (2009) Brassica shatter-resistance research update. In,     16th Australian Research Assembly on Brassicas. Ballarat, Victoria,     2009 -   Chalhoub, B. et al., (2014) Early allopolyploid evolution in the     post-Neolithic Brassica napus oilseed genome. Science 345, 950-953.

Example 2: Generation of Shatterproof Gene Knock-Out Lines in the BN2-SU-H Canola Line Using CRISPR/Cas9 Plasmid Delivery into Protoplasts

In this Example, sulphonylurea-tolerant canola plant lines with non-functional (KO) Shatterproof (SHP) genes using CRISPR/Cas9 were generated. The CRISPR/Cas9 gene and sgRNAs contained within plasmids were delivered to protoplasts isolated from leaves of haploid plants to knock down the BnSHP genes. The sgRNA is a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNA guides the Cas9 to the target genes, where Cas9 makes a double-stranded break in each of the BnSHP gene in a site-directed manner. The double-stranded breaks in the BnSHP gene when repaired by the ubiquitous, error-prone NHEJ pathway will cause InDels (nucleotide Insertions or Deletions) to form around the cleavage site. Loss of function alleles of the BnSHP genes occur when these InDels created by Cas9 shift the reading frame of the SHP genes.

The percentage of InDel formation in each of the 8 SHP gene targets in shoots regenerated from protoplasts treated with CRISPR/Cas9 was between 20 to 40%, as determined by Next Generation Sequencing (FIG. 3 ). The most common InDels identified were +1 insertions (up to 10%), and −1 and −2 deletions (5% and 1.5% respectively; data not shown). Shoots with mutations in 1 through 8 of the genes were identified with different frequencies (Table 2). Approximately 70% of the shoots contained InDels in at least one SHP gene. Sequence analysis of the target region (around the Cas9 cleavage site) in each gene indicated that not all of the InDels in these genes resulted in a non-functional (KO) allele due to a frame shift. The majority of the plants had between 2 and 5 gene KOs (Table 2). Out of this experiment, 80 independent plant lines were regenerated containing InDels in 2 to 8 of the BnSHP genes, showing that Cas9 is active and able to cleave all 8 target SHP genes in B. napus and form InDels. Moreover, these InDels can generate non-functional gene KOs after the CRISPR/Cas9 plasmid was introduced, by shifting the reading frame of the SHP genes.

TABLE 2 Frequency of BnSHP genes with InDels and non-functional gene KOs in regenerated shoots screened by NGS SHP Genes Shoots SHP Gene Shoots with InDels # % KOs* # % 0 133 29 0 149 33 1 40 9 1 36 8 2 38 8 2 62 14 3 49 11 3 80 18 4 57 12 4 62 14 5 50 11 5 40 9 6 57 12 6 14 3 7 19 3 7 8 2 8 11 2 8 3 1 Total Shoots 454 100 Total Shoots 454 100 *Number of non-functional alleles of BnSHP genes due to shift in the reading frame caused by InDels.

Methods

Canola protoplasts were isolated from leaves of in vitro-grown BN2-SU haploid plants derived from microspore culture (Sun et al., 1998; Swanson, E., 1990). The CRISPR/Cas9 encoded plasmids contains pMas::Cas9 with a pea rbcSE9 terminator and AtU6P::sgRNA with a poly-T₁₀ terminator. Sequences of features are as follows: amino acid sequence of Cas9 (SEQ ID NO: 50), nucleotide sequence of Mas promoter (SEQ ID NO: 51), nucleotide sequence of rbcSE9 terminator (SEQ ID NO: 52), nucleotide sequence of AtU6 promoter (SEQ ID NO: 53), and nucleotide sequence of poly-T₁₀ terminator (SEQ ID NO: 54).

The CRISPR/Cas9 plasmids were introduced into protoplasts by PEG mediated delivery at a final concentration of 0.05 μg/μL. Protoplasts were cultured in liquid medium (2.5×10⁵ cells/mL) and incubated in the dark at 25° C. Cell samples were obtained after one week and analyzed by NGS. After 6-8 weeks, protoplast-derived microcalli were plated over solid regeneration medium, and shoots started differentiating from regenerated calli after about 2-4 weeks. Leaf samples from fully differentiated shoots were analyzed by NGS to determine the occurrence of InDels in targeted SHP genes. Elongated shoots were micropropagated and rooted plants were transferred to soil and hardened in a growth chamber for 2-4 weeks until the plants were well established.

The CRISPR/Cas9 consists of two components: the plant codon-optimized Streptococcus pyogenes Cas9 (SpCas9) and sgRNAs were expressed from separated plasmids. The sgRNA is a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNA region contains the spacer sequences described in Table 3, which were used to guide the Cas9 nuclease to the target gene. In this experiment the CRISPR/Cas9 targets the BnSHP genes.

TABLE 3 gRNAs targeting BnSHP genes BnSHP  gRNA  gRNA Sequence  Gene  Name (5′ to 3′) Target BnSHP-2 GTAGCAAGAAGATAGGTAGA  1A, 1C (SEQ ID NO: 42) BnSHP-3 GTAACAAGAAGCTAGTGAGA  3A, 3C (SEQ ID NO: 43) BnSHP-4 GTAGCAAGAAGCTAGTAAGA  2A, 2C,  (SEQ ID NO: 44) 3A, 3C BnSHP-5 GCAGCAAGAAGATAGGGAGA  4A, 4C (SEQ ID NO: 45) BnSHP-9 CAGAAGCAATGGATGAAGGT  1A, 1C (SEQ ID NO: 46) BnSHP-10 CAGAATCAATGGAGGAAGGT  2A, 2C,  (SEQ ID NO: 47) 3A, 3C BnSHP-11 GGGTTGATATAAATGGAGGG  4A, 4C (SEQ ID NO: 48) BnSHP-12 CAGAAGCAATGGATGAAAGT  1A, 1C (SEQ ID NO: 49)

REFERENCES

-   Sun et al., “Cotyledon-derived diploid and haploid protoplast     culture and diploid plant regeneration in Brassica napus cv.     ‘Topas’,” Can. J. Bot. 76: 530-541, 1998. -   Swanson, E., “Microspore Culture in Brassica,” Methods in Molecular     Biology, Vol. 6, Chapter 17, p. 159-69, 1990.

Example 3: Generation of Shatterproof Gene Knock-Out Canola Lines by Delivering CRISPR/Cas9 Ribonucleoprotein (RNP) to Protoplasts

Similar to the previous Example 2, the purpose of this Example is to generate SU-tolerant canola plant lines with non-functional (KO) Shatterproof (SHP) genes using CRISPR/Cas9. The CRISPR/Cas9 used to knock down the BnSHP genes in the B. napus genome are delivered to leaf derived protoplasts as Cas9 protein complexed with gRNAs (RNPs), in combination with single-stranded oligonucleotides (ssODNs or GRONs, Table 4) targeting three specific InDel mutations (+1 insertion, −1, and −2 deletions) to disrupt the function of each of the 8 BnSHP paralogous genes. Before delivery to protoplasts, recombinant Cas9 protein (commercially available) is complexed in vitro with the gRNA (Table 5), which is in vitro synthesized from a plasmid DNA template to generate a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Once within the cells, the crRNA guides the Cas9 to the target genes, where Cas9 makes a double-strand break in each of the BnSHP gene in a site-directed manner. In the presence of the GRONs, the double-stranded breaks in the BnSHP genes could be repaired by the Homologous Direct Repair (HDR) pathway, in addition to the NHEJ pathway. The HDR pathway can use the GRONs as DNA templates, introducing targeted mutations specified in the GRON sequences (i.e., n+1, n−1, and n−2). Loss of function alleles of the BnSHP genes occur when these InDels shift the reading frame of the SHP genes, leading to truncated and non-functional gene products (mRNA and proteins, Table 6).

Results

InDel formation in 1 and up to 8 of the SHP genes were observed in over 95% of the shoots regenerated from protoplasts treated with CRISPR/Cas9, as determined by Next Generation Sequencing. GRON targeted mutations (+1, −1, −2 nucleotide insertion or deletions) were found in over 90% of the shoots with InDels in SHP genes. Shoots with mutations in each of the 8 SHP genes, and with combinations of multiple gene KOs were identified with different frequencies. Out of 5395 shoots screened from three consecutive experiments, 1127 independent plant lines were regenerated containing targeted InDels in 1 to 8 of the BnSHP genes, including a total of 153 unique KO genotypes (out of 255 possible). Plant KO lines representing each of the SHP KO genotypes were successfully transferred to soil and grown to maturity in the greenhouse for phenotypic analyses.

Methods

Canola protoplasts are isolated from leaves of in vitro microprogated haploid plants. Cas9 protein complexed with gRNAs (Table 5), along with single-stranded oligonucleotides (ssODNs; GRONs) make precise gene specific mutations in each of the 8 BnSHP paralogous genes (Table 4). Cas9 protein complexed with gRNAs and GRONs is introduced into protoplasts by PEG mediated delivery at a final concentration of 0.05 μg/μL and 0.5 μM, respectively. Protoplasts are cultured in liquid medium (1.25×10⁵ cells/mL) and incubated in the dark at 25° C. Cell samples are obtained after one week and analyzed by NGS. After 6-8 weeks, protoplast-derived microcalli are transferred to solid regeneration medium, and shoots start differentiating from regenerated calli after about 2-4 weeks. Leaf samples from fully differentiated shoots are analyzed by NGS to determine the occurrence of InDels in targeted SHP genes. Elongated shoots are micropropagated, and rooted plants are transferred to soil and hardened in a growth chamber for 2-4 weeks until the plants are well established.

The GRONs used with the Cas9 RNP contain the coding sequence of the targeted SHP genes around the site of conversion and are labeled with a 2′-O-Me group at the first 5′ base of the GRON which is an RNA base instead of a DNA base (Table 4). The CRISPR/Cas9 consists of two components: the plant codon-optimized Streptococcus pyogenes Cas9 (SpCas9) and sgRNAs are expressed as protein and RNA respectively. The sgRNA is in vitro transcribed from a DNA template, and it is a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNA region contains the spacer sequences described in Table 5, which are used to guide the Cas9 nuclease protein to the target gene. In this Example the CRISPR/Cas9 targets the BnSHP genes.

TABLE 4 Sequence of ssODNs (GRONs) ID Sequence (5′ to 3′) BnSHP(-1;CR2-5)/ A*GAGAGYARCAAGAAGMTAG C/41 KAGAGGGAAGATAGAGATAAA (SEQ ID NO: 55) BnSHP(-2R;CR2-5)/ A*GAGAGYARCAAGAAGMTAG C/41 AGAGGGAAGATAGAGATAAAG (SEQ ID NO: 56) BnSHP(+1;CR2-5)/ A*GAGAGYARCAAGAAGMTAGK C/42 DNAGAGGGAAGATAGAGATAAA (SEQ ID NO: 57)

TABLE 5 gRNAs targeting BnSHP genes BnSHP  gRNA gRNA Sequence  Gene  Name (5′ to 3′) Target BnSHP-2 GTAGCAAGAAGATAGGTAGA  1A, 1C (SEQ ID NO: 42) BnSHP-3 GTAACAAGAAGCTAGTGAGA  3A, 3C (SEQ ID NO: 43) BnSHP-4 GTAGCAAGAAGCTAGTAAGA  2A, 2C,  (SEQ ID NO: 44) 3A, 3C BnSHP-5 GCAGCAAGAAGATAGGGAGA  4A, 4C (SEQ ID NO: 45)

TABLE 6 Size of predicted truncated, non-functional protein products generated by the introduction of targeted mutation n − 1, n − 2, and n + 1 (A, G, C, and T) in each of the SHP genes Stop Codon (AA#) SHP n + 1 n + 1 Gene WT Reference n − 2 n − 1 (A, G, C) (T) 1A 276 48 21 49 18 1C 136 48 21 49 18 2A 250 37 21 38 18 2C 249 37 21 38 18 3A 277 48 21 49 18 3C 276 48 21 49 18 4A 245 48 21 49 18 4C 245 44 21 45 18

Example 4: Phenotyping of Shatterproof Mutant Canola Lines

In Arabidopsis thaliana, SHATTERPROOF 1 AND 2 (SHP1/SHP2) are transcription factors members of the MADS-box family involved in the formation of the dehiscent zone (DZ), a layer of cells between fruit valves responsible for the shatter of mature pods (Liljegren et al., 2000; Roeder and Yanofsky, 2006). The differentiation of the DZ in developing pods is characterized by the formation of a layer of cells with lignified cell walls, and a separation cell layer. Arabidopsis thaliana shp1 shp2 double mutants fail to develop a functional DZ, which does not lignify or has a defined separation layer, and, as a consequence, the fruits are indehiscent and do not open at the end of development. In canola (Brassica napus), Applicant has identified and characterized 8 genes that are highly homologous to the Arabidopsis SHP1/SHP2. The BnSHP genes also appear to be involved in the differentiation of the DZ and play a similar role in controlling the shattering of mature pods in canola.

Results

Loss-of-function studies indicate that SHP1 and SHP2 promote lignification of a subset of valve margin cells in Arabidopsis fruit. The lignification patterns of fruits obtained from different BnSHP KO C₀ lines were analyzed and compared to wild-type fruit. There is a clear reduction in valve margin cell lignification in fruits of BnSHP KO lines (FIG. 4 ). In mutated lines with 7-8 BnSHP gene KOs, no valve margin lignification was observed at the base of fruits, whereas lignified valve margin cells stained with phloroglucinol are present in wild type fruits, as well as in fruit from mutant plant lines with low number of gene KOs (FIG. 4 ).

A pod breaking test using a TissueLyser and a Geno/Grinder was also used to assess shatter resistance in mature dried pods. The two tests show a correlation between the number of BnSHP gene KOs and pod shatter reduction (FIG. 5 ). A correlation (r=0.86) is also found between shaking frequency to pod shatter reduction and staining score of lignified layers (Table 7A). A separate experiment with different KO lines showed similar results (Table 7B).

TABLE 7A Comparison of pod shattering test and lignified layer staining results— Experiment 1 Staining Score Shaking of Lignified Frequency to Gene KO Candidate Line ID Layer (C₀) Shatter (C₁) Description Genotype Control (WT) 1 16 N/A N/A A02-0230 1 18 2 KO 4A/4C A01-1013 2 16 2 KO 1C/2C A01-0151 1 16 3 KO 2A/3A/4C A02-0232 1 20 4 KO 1A/1C/4A/4C A01-0037 1 18 4 KO 1C/2A/3C/4C A01-0164 4 18 6 KO 1A/2A/3A/3C/4A/4C A01-1315 1 20 6 KO 1C/2C/3A/3C/4A/4C A01-0069 4 30 7 KO 1C/2A/2C/3A/3C/4A/4C A01-1291 4 24 7 KO 1C/2A/2C/3A/3C/4A/4C A01-1166 3 30 7 KO 1A/2A/2C/3A/3C/4A/4C A01-0222 5 30 8 KO 1A/1C/2A/2C/3A/3C/4A/4C A01-1187 5 30 8 KO 1A/1C/2A/2C/3A/3C/4A/4C A01-0025 4 30 8 KO 1A/1C/2A/2C/3A/3C/4A/4C A01-0022 4 30 8 KO 1A/1C/2A/2C/3A/3C/4A/4C

TABLE 7B Comparison of Pod shattering test and lignified layer staining results— Experiment 2 Staining Score Shaking of Lignified Frequency to Gene KO Genotype Line ID Layer (C₀) Shatter (C₀) Description (SHP Gene KO) Control (WT) 1 14.0 N/A N/A A05_1098 1 14.3 1 KO 1A A05_1884 1 15.3 1 KO 4C A05_0085 1 15.0 2 KO 1A/3A A05_0641 1 14.3 2 KO 3C/4A A05_1094 1 14.0 2 KO 3A/3C A05_0375 1 14.0 3 KO 1C/3A/3C A05_2790 1 14.5 3 KO 2A/4A/4C A05_0102 2 16.0 4 KO 3A/3C/4A/4C A05_0129 1 16.0 4 KO 1C/3A/3C/4A A05_0415 1 16.3 4 KO 1C/2A/2C/3C A06_0387 1 16.5 4 KO 2A/3A/3C/4C A08_0780 1 15.3 4 KO 1C/2C/3C/4A A05_0751 3 18.3 5 KO 1C/2A/3A/3C/4A A05_1608 4 19.0 5 KO 2A/3A/3C/4A/4C A05_1894 2 16.5 5 KO 2A/2C/3A/3C/4C A08_0068 2 17.0 5 KO 1A/2C/3C/4A/4C A05_0277 2 18.0 6 KO 1C/2A/2C/3A/3C/4A A05_1217 2 17.0 6 KO 1C/2A/2C/3A/3C/4C A05_0342 3 18.0 7 KO 1A/1C/2A/2C/3A/3C/4A A05_1635 4 24.0 7 KO 1A/1C/2A/2C/3A/4A/4C A05_3484 3 22.0 7 KO 1A/1C/2A/2C/3A/3C/4C

Methods

Phloroglucinol staining of lignified cell layer in the valve dehiscent zone. Developing fruit from wild-type BN2-SU and different SHP KO lines (C₀ plants) are collected to examine the lignification pattern of fruits. For lignin staining, cross sections of fruits are obtained with a razor blade and the sections are stained for 2 min in a 2% phloroglucinol solution in 95% ethanol, then photographed in 66% perchloric acid. The intensity of the phloroglucinol staining of the lignified cell layer correlates with the amount of lignin in the cell walls (Liljegren et al., 2000; Roeder and Yanofsky, 2006), and it is scored as shown in Table 8 below.

TABLE 8 Lignification score summary Phloroglucinol staining result Score Very dark stained lignified layer 1 Medium dark stained lignified layer 2 Light stained lignified layer 3 Very light stained, Partial absent the lignified layer 4 Totally absent lignified layer 5

Pod shattering test using a TissueLyser. Candidate lines are transplanted into 3.5″ pots using Sunshine Mix 4 media and kept under T12 fluorescent grow lights at a 14-hour photoperiod. After three weeks the lines are transplanted into 5.5″ pots and moved the greenhouse, where the maximum cooling temperature is set at 78° F. Plants are grown under standard canola maintenance conditions and perforated pollen bags are employed to prevent outcrossing and contamination. The plants are taken off water at a 30% seed color change, and continued to dry down in the greenhouse until the seeds reach a 100% color change, when the plants are fully desiccated. The pods are collected and placed in an oven at 30° C. for 1h to ensure uniform levels of moisture across all samples.

The following candidate lines are selected to phenotype using the shatterproof breakage test (See also FIG. 5 ): BN2 Diploid WT (undried), BN2 Diploid WT (dried), SHP-A01-0151 (2 KO), SHP-A01-0037 (3 KO), SHP-A01-1098 (5 KO), SHP-A01-0154 (6 KO), SHP-A01-0222 (7 KO).

The shatterproof phenotype is determined by the level of valve separation found under controlled agitation of the pods. To test the valve separation, single pods are placed into a 96 well deep trough container and secured in the arms of a TissueLyser II (Qiagen, Germany). The single pod samples are run on the TissueLyser for 30 seconds at frequencies of 22, 23, 24, 25, 26, 27, 28, 29, and 30 Hz. Per line, four single pods reps were tested at each frequency. The phenotype was scored on a scale of 1-4. An intact pod was given a score of one, a partially split pod with connected valves was scored a two, a score of three represented the separation of one valve, and a score of four indicated that both valves were separated from the replum.

Pod shattering test of C₁ generation of KO lines. Canola seeds (C₁) are germinated in plugs, where they remain for 3 weeks. Plants are then transferred into 4″ pots and moved to the greenhouse under 14-h photoperiod with 23-25° C. Day/19-21° C. Night temperatures. Pods are collected from fully mature plants, and then dried in plastic containers with holes for about 3 weeks. The pods are completely dry before testing. Three uniform pods are chosen and placed in 3 separated cells of a modified shaking box. The box is then loaded onto a TissueLyser II. The frequency of shaking is set to a certain frequency setting (starting from 12 Hz), with a shaking time set to 30 seconds. When the shaking stopped, the box is unloaded, and pods are evaluated using a shattering score. Each frequency setting is tested on 12 pods per plant. The average score from the 12 pods is used as the final score for each frequency. When the shaking score is greater than 2.5, the corresponding frequency represents the pod shattering frequency set point.

Pod shattering test using the Geno/Grinder 2010 (SPEX Sample Prep, USA). Candidate lines are transplanted into 3.5″ pots using Sunshine Mix 1 media and kept under T12 growth lights at a 16h photoperiod and 21° C./19° C. day/night temperature for hardening. After three weeks, the lines are transplanted into 5.5″ pots and moved the greenhouse, where the maximum cooling temperature is set at 78° F. Plants are grown under standard canola maintenance conditions and perforated pollen bags are employed to prevent outcrossing and contamination. The plants are taken off water at a 30% seed color change and continue to dry down in the greenhouse until the seeds reach a 100% color change and the plants are fully desiccated. The pods from each individual plant are collected in a plastic container with a cover with a hole in the center, which is placed in an oven at 40° C. for at least 12 h to ensure uniform levels of moisture across all samples.

The shatterproof phenotype is determined by the level of valve separation found under controlled agitation of the pods. To test the valve separation, 12-24 pods are placed into a 96 well deep trough container and secured in the arms of a Geno/Grinder 2010 (SPEX Sample Prep, USA). The containers holding pod samples are run for 20 sec at different rpm (for example at 720, 750, 780, 810, 840, 870, 900, 930, 960, 990, 1020, 1050, 1080 rpm). At the end of the run, the container is taken off the machine and the shattering score is given to each pod according to the score table (Table 9). When the average shattering score under the certain rpm is greater than 2.5, the rpm value will be the pod shattering value for the line. This method can handle more pods at one time and is much faster to run than the TissueLyser test. In order to validate the Geno/Grinder Method, the negative check and positive checks, along with a few lines were run using both TissueLyser and Geno/Grinder. The corresponding shattering scores were collected. The R value of the both data sets is 0.88, which indicates the data collected using both methods are highly correlated (FIG. 6 ).

TABLE 9 Shattering Score Summary Shaking Assay Result Score Intact pod that has no cracks or damage visible 1 Pod has a small crack at the end, no seeds have 2 escaped. There is a visible crack that is greater than 3 half the pod, the two valves are still attached to the septum. Seeds might have escaped One of the valves has separated from the septum. 4 Seeds have escaped Both valves have separated from septum and pod 5 has fully shattered.

Example 5: Generation and Field Testing of Shatterproof KO Lines

This Example shows the evaluation and selection of top performing SHP KO lines. Since pod shattering traits are controlled by multiple genes and could be affected by environmental factors (e.g., biotic and abiotic), it is important to evaluate the pod shattering trait in multiple environments and years.

Results

Pod shatter resistance of SHP KO lines was evaluated first in the greenhouse (C₀ generation), and then in two different locations over a two-year period (C₁ and C₂ generations). Selected lines with 5 to 8 SHP gene KOs consistently showed much better performance than the negative WT control (FIG. 7 ). The KO lines also showed similar or better pod shatter reduction phenotypes compared to the commercial shatterproof lines checks (positive controls). For example, A05_1635, A05_0342, A05_0277, A05_2013 and A05_0071 were very stable, and they did not show any abnormal growth phenotype in any of the tested environments. These KO lines could be considered true shatterproof lines.

The SHP genes (either mutant or wild type) in each line were sequenced to confirm presence of the mutation in the respective SHP gene where applicable. The sequence of the full target amplicon of the area around the target region is presented in Table 10 below.

TABLE 10 SHP Sequences in Mutant Lines SHP Gene SEQ ID NO: Line 1A 1C 2A 2C 3A 3C 4A 4C A05_0071 62 72 82 92 102 112 122 132 A05_2013 63 73 83 93 103 113 123 133 A05_1635 64 74 84 94 104 114 124 134 A05_0342 65 75 85 95 105 115 125 135 A05_0113 66 76 86 96 106 116 126 136 A05_0277 67 77 87 97 107 117 127 137 A05_0272 68 78 88 98 108 118 128 138 A05_1600 69 79 89 99 109 119 129 139 A05_0751 70 80 90 100 110 120 130 140 A05_1894 71 81 91 101 111 121 131 141

Methods

CRISPR/Cas9 protein complexed with gRNAs (RNPs, Table 5), along with single-stranded oligonucleotides (GRONs) (Table 4) are used to knock out BnSHP genes in an otherwise wild-type background canola line. The CRISPR/Cas9 consists of two components: the plant codon-optimized Streptococcus pyogenes Cas9 (SpCas9) and sgRNAs that are expressed as protein and RNA respectively. The sgRNA is in vitro transcribed from a DNA template, and it is a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNA region contains the spacer sequences described in Table 5, which are used to guide the Cas9 nuclease protein to each of the target SHP genes. The GRONs contain the coding sequence of the targeted SHP genes around the site of conversion, carry precise gene specific mutations (n+1, n−1, and n−2), and are labeled with a 2′-O-Me group at the first 5′ base, which is a RNA base instead of a DNA base (Table 4).

RNPs and GRONs were introduced into protoplasts by PEG mediated delivery at a final concentration of 1.0 μg/μL and 0.05 μM, respectively. Before delivery to protoplasts, the recombinant Cas9 protein was complexed in vitro with the gRNA. Canola protoplasts were isolated from leaves of in vitro micropropagated plants, following a standard protocol. Protoplasts were cultured in liquid medium (1.25×10⁵ cells/mL) and incubated in the dark at 25° C. Cell samples were obtained after one or three weeks, and analyzed by deep sequencing, to determine the frequency of mutations in target genes. After 6-8 weeks, protoplast-derived microcalli were transferred to solid regeneration medium, and shoots started differentiating from regenerated calli after about 2-4 weeks. Leaf samples from fully differentiated shoots were analyzed by NGS to determine the occurrence of targeted mutations in each of the 8 SHP genes. Shoots with targeted mutations in individual and multiple genes, covering all 255 possible gene KO combinations or genotypes were then screened for ploidy. Diploid plants were micropropagated in vitro, and transferred to soil in a growth chamber. Hardened (C₀) plants were transferred to the greenhouse and grown to maturation (seed setting).

Seeds harvested from C₀ plants are called C₁ generation. At least 3 C₀ plants for each KO combination were selected and grown as stated in Example 4. During the hardening process, leaf samples were collected and the genotypes of C₀ plants were confirmed by NGS. C₁ seeds were germinated in plugs (5 plants per line), and leaf samples were collected 10-12 days after planting for genotype confirmation. Three weeks after planting, the C₁ plants were transferred into 5.5″ pots and moved to the greenhouse, where the maximum cooling temperature is set at 78° F. Plants were grown under standard canola maintenance conditions and perforated pollen bags were employed to prevent outcrossing and contamination. The plants were taken off water at a 30% seed color change and continued to dry down in the greenhouse until the seeds reached a 100% color change and the plants were fully desiccated. The pods from either C₀ plants or C₁ plants grown in the greenhouse were collected and placed in an oven at 40° C. for at least 12 h to ensure uniform levels of moisture across all samples. Pod shattering was evaluated using the TissueLyser Method. The selected lines were also tested under field conditions in two different locations: one in California and one in North Dakota. A randomized complete block design (RCBD) with three replications was used to design the experiments. C₁ and C₂ seeds were treated with fungicide (Helix, Bayer Crop Science) and directly sowed in the soil. Under the growing environments, plants grew to maturity, and the pods were collected as a bulk sample for each line from each replication. Pod shattering phenotypes were evaluated using either the TissueLyser or the Geno/Grinder. The data were collected and analyzed.

REFERENCES

-   Bohanec B (2003) Ploidy determination using flow cytometry. In:     Maluszynski M, Kasha K J, Forster B P, Szarejko I (eds) Doubled     haploid production in crop plants: a manual. Kluwer, Dordrechts, pp     397-403. 

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 47. A plant or part thereof comprising at least one mutation in at least three, at least four, at least five, at least six, at least seven, or eight nucleic acid sequences encoding SHATTERPROOF (SHP) genes.
 48. The plant or part thereof of claim 47, wherein the nucleic acid sequences have at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to nucleic acid sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:
 8. 49. The plant or part thereof of claim 47, wherein the nucleic acid sequences are selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:
 8. 50. The plant or part thereof of claim 47, wherein the mutation is a frameshift mutation.
 51. The plant or part thereof of claim 50, wherein the frameshift mutation results in one or more nucleotide insertions or deletions as compared to the corresponding endogenous gene without the frameshift mutation.
 52. The plant or part thereof of claim 50, wherein the frameshift mutation results in a premature stop codon.
 53. The plant or part thereof of claim 50, wherein the mutation reduces or eliminates the expression or activity of the SHP gene and/or SHP polypeptide.
 54. The plant or part thereof of claim 47, wherein the plant exhibits reduced susceptibility to preharvest dehiscence.
 55. The plant of claim 47, wherein the plant is selected from the group consisting of Brassica napus, Brassica rapa, Brassica oleracea, Brassica juncea, Brassica species, Raphanus sativus, Pisum sativum, Phaseolus vulgaris, Lens culinaris, Glycine max, and Fabaceae species.
 56. A method of producing the plant of claim 47, comprising the steps of: a) introducing mutations into plant cells, wherein the mutations are at least one mutation in at least three, at least four, at least five, at least six, at least seven, or eight nucleic acid sequences encoding SHP genes; b) selecting plant cells containing the mutations; and c) regenerating a plant having the mutations; wherein the plant exhibits reduced susceptibility to preharvest dehiscence.
 57. The method of claim 56, wherein the mutations are introduced using one or more vectors, wherein the vectors comprise gene editing components selected from the group consisting of a nuclease, an RNA-guided DNA endonuclease, a CRISPR/Cas9 system, a TALEN, a zinc finger, and a meganuclease designed to target a nucleic acid sequence encoding a SHP gene.
 58. The method of claim 56, wherein the mutations are introduced using a GRON system designed to target a nucleic acid sequence encoding a SHP gene.
 59. The method of claim 58, wherein the GRON system comprises one or more modifications selected from the group consisting of a Cy3 group, 3PS group, and a 2′O-methyl group.
 60. The method of claim 56, wherein the nucleic acid sequences have at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to nucleic acid sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:
 8. 61. The method of claim 56, wherein the nucleic acid sequences are selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:
 8. 62. The method of claim 56, wherein the mutation is selected from the group consisting of a frameshift mutation, a frameshift mutation resulting in one or more nucleotide insertions or deletions as compared to the corresponding endogenous gene without the frameshift mutation, and a frameshift mutation resulting in a premature stop codon, and wherein the mutation reduces or eliminates expression of the SHP gene and/or SHP polypeptide.
 63. The method of claim 56, wherein the plant is selected from the group consisting of Brassica napus, Brassica rapa, Brassica oleracea, Brassica juncea, Brassica species, Raphanus sativus, Pisum sativum, Phaseolus vulgaris, Lens culinaris, Glycine max, and Fabaceae species.
 64. An F1 plant, wherein the F1 plant has the plant of claim 47 as a parent.
 65. A method of making plant seeds, the method comprising crossing the plant of claim 47 with another plant and harvesting seed therefrom.
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 67. A plant produced by growing the seed of claim 65, wherein the plant has all the physiological and morphological characteristics of a plant comprising at least one mutation in at least three, at least four, at least five, at least six, at least seven, or eight nucleic acid sequences encoding SHATTERPROOF (SHP) genes. 